Xiaoyun Pu

Procalcitonin sensitive detection based on graphene–gold nanocomposite film sensor platform and single-walled carbon nanohorns/hollow Pt chains complex as signal tags

Septicemia is a serious disease that requires early diagnosis, and procalcitonin (PCT) serves as a diagnostic biomarker for this disease. Traditional clinical detection (via immune-gold chips) remains difficult and expensive. An electrochemical immunosensor based on new nanomaterials may provide a solving approach. Herein, an ultrasensitive sandwich electrochemical strategy for PCT detection was developed. Firstly, reduced graphene oxide (rGO)-gold (Au) nano-composite film was used as the immunosensor platform to increase the amount of PCT antibody 1(Ab1) immobilized. Next, single-walled carbon nanohorns (SWCNHs)/hollow Pt chains (HPtCs) complex was firstly utilized to label PCT Ab2 as signal tags. For SWCNHs with few side effects, high surface area and HPtCs with higher specific surface, better catalytic activity, complex synthesized from both may provide more advantages. Moreover, to amplify signal, HPtC catalytic activity with H2O2 was enhanced by horseradish peroxidase (HRP) for dual synergy amplification. The whole results demonstrated that the proposed immunosensor exhibited fast operation, high sensitivity, good reproducibility, acceptable stability and ideal selectivity compared with traditional method. The linear calibration of the immunosensor ranged from 1.00 pg/mL to 2.00 × 10(1)ng/mL with a detection limit of 0.43 pg/mL. Analytical application results revealed that the immunosensor matched with the real concentrations of serum samples. Overall this immunosensor may provide a new alternative strategy for PCT detection.

Identification of Endogenous Controls for Analyzing Serum Exosomal miRNA in Patients with Hepatitis B or Hepatocellular Carcinoma

Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for diagnosing cancer. The canonical technique for measuring miRNA transcript levels is reverse transcription quantitative polymerase chain reaction (RT-qPCR). One prerequisite for validating RT-qPCR data is proper normalization with respect to stably expressed endogenous reference genes. However, genes that meet all of the criteria of a control gene for exosomal miRNAs have not yet been identified. To find out the control gene for exosomal miRNAs, we evaluated the expression stability of 11 well-known reference genes in circulating exosomes. In this study, we found that the combination of miR-221, miR-191, let-7a, miR-181a, and miR-26a can be an optimal gene reference set for normalizing the expression of liver-specific miRNAs. This combination enhanced the robustness of the relative quantification analyses. These findings highlight the importance of validating reference genes before quantifying target miRNAs. Furthermore, our findings will improve studies that monitor hepatitis progression and will aid in the discovery of noninvasive biomarkers to diagnose early stage HCC.

Ultrasensitive strategy based on PtPd nanodendrite/nano-flower-like@GO signal amplification for the detection of long non-coding RNA

Highly up-regulated in liver cancer (HULC) is a novel promising noninvasive biomarker for hepatocellular carcinoma (HCC), which is a kind of long non-coding RNAs (lncRNAs). But traditional methods limited HULC clinical detection for ownself drawbacks. Development a new HULC detection approach is urgent and necessary. Electrochemical nucleic acid sensor based on different signal amplification strategies with high sensitivity, fast, simple, and convenient, may solve this problem. Herein, we propose a novel strategy based on Pt-Pd bimetallic nanodendrites/nanoflower-like clusters on graphene oxide/Au/horseradish peroxidase (PtPd BND/BNF@GO/Au/HRP) to enhance the catalytic efficiency and sensitivity. And Au particles were simultaneously and separately capped with thionine or detection probe, which increase the binding amount of detection probe and decrease the electronic background. The results indicated that the catalytic effect was noticeably elevated and that the biosensor provides ultrasensitive detection for the lncRNA HULC. The linear calibration of the biosensor ranged from 1.00×10(-3) to 1.00×10(3) pM/mL, and the limit of detection was 0.247 fM/mL. The lncRNA biosensor based on the PtPd BND/BNF@GO/Au/HRP/Au/thionine exhibited acceptable reproducibility and clear selectivity. This strategy may provide a new alternative for clinical HCC diagnosis through the detection of HULC.

GLIPR-2 Overexpression in HK-2 Cells Promotes Cell EMT and Migration through ERK1/2 Activation

The epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in the adult kidney is one of the key events in renal interstitial fibrosis. Glioma pathogenesis related-2 (GLIPR-2) has been shown to be up-regulated in proximal tubular cells (PTCs) in the fibrotic kidney. However, the biological function of GLIPR-2 remains unknown. In this study, we found that GLIPR-2 expression is elevated in the kidney tissue samples of patients with diabetic nephropathy (DN). Human proximal renal tubular epithelial cells (HK-2 cells) were transfected with pcDNA3.0-GLIPR-2 and selected with G418. To identify the biological function of GLIPR-2, an epithelial-to-mesenchymal transition (EMT) PCR array analysis was performed, and genes that had statistically significantly altered expression levels with more than a two-fold difference compared with the pcDNA3.0-transfected HK-2 cells were considered. Key elements of the EMT process, such as E-cadherin and vimentin, were transcriptionally activated in the pcDNA3.0-GLIPR-2-transfected sublines. In addition, α-SMA gene expression, which is a marker of myofibroblasts, increased in the pcDNA3.0-GLIPR-2-transfected HK-2 cells. The cell migration assay demonstrated that the transfection of HK-2 with GLIPR-2 promoted cell migration following an EMT. Additionally, consistent with the effects of increased EGFR expression levels, we found that the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was highly elevated in the pcDNA3.0-GLIPR-2-transfected group. Our study demonstrates that GLIPR-2 overexpression in HK-2 cells can potentiate EMT-like processes in this cell type through the ERK1/2 signaling pathway. GLIPR-2 may be responsible for the development of renal fibrosis by increasing the accumulation of interstitial fibroblasts.

Hypoxia Promotes Epithelial - Mesenchymal Transition of Hepatocellular Carcinoma Cells via Inducing GLIPR-2 Expression

Glioma pathogenesis related-2 (GLIPR-2) belongs to pathogenesis related-1 (PR-1) family whose function remains unknown. In our previous studies, GLIPR-2 was found to be a novel potent stimulator of epithelial-to-mesenchymal transition (EMT) in renal fibrosis which has been classified as type 2 EMT. However, whether GLIPR-2 could induce type 3 EMT in carcinogenesis needs further investigation. In this study, we showed that GLIPR-2 was expressed in hepatocellular carcinoma (HCC) tissues, hypoxia could upregulate the expression of GLIPR-2 in HepG2 and PLC/PRF/5 cells in vitro, overexpression of this protein promoted migration and invasion via EMT, knockdown of GLIPR-2 attenuated migration and invasion of HepG2 and PLC/PRF/5 cells in hypoxia. Moreover, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are positively regulated by GLIPR-2. Taken together, we provide evidence for a hypoxia/GLIPR-2/EMT/migration and invasion axis in HCC cells and it provides novel insights into the mechanism of migration and invasion of hepatocellular carcinoma cells in hypoxia condition.

Induction of an electrochemiluminescence sensor for DNA detection of Clostridium perfringens based on rolling circle amplification

Clostridium perfringens is one of the predominant pathogens causing infectious diseases. This work describes the application of a rolling circle amplification (RCA) based electrochemiluminescence sensor for detection of C. perfringens. Firstly, the target DNA is captured by the probes on the pretreated electrode. Subsequently, RCA reaction is executed isothermally. The products of RCA are incubated with hemin, resulting in the decrease of the ECL emission, which is related to the quantity of the target DNA. The ECL-sensor provides the capability of discriminating the target DNA from non-target sequences even with only one base difference, suggesting an advantageous selectivity. Meanwhile, the lowest concentration of the target DNA is 10−15 M, showing satisfactory sensitivity. Therefore, this strategy combines amplification ability of RCA and high sensitivity of ECL, and enables a low fM detection of C. perfringens without the bacterial culture.

Development of a real-time resistance measurement for Vibrio parahaemolyticus detection by the lecithin-dependent hemolysin gene.

The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg2+), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg2+) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1–2 hours, with a minimum bacterial density of 10 CFU.mL−1 and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.

A novel strategy of procalcitonin detection based on multi-nanomaterials of single-walled carbon nanohorns–hollow Pt nanospheres/PAMAM as signal tags

Procalcitonin (PCT) is a common clinical biomarker of septicemia, and precise detection of PCT is critical to the treatment of septicemia. Conventional detection methods suffer various shortcomings that hinder precise PCT detection. An electrochemical immunosensor based on the antigen–antibody immune-reaction may provide a solution. We developed a novel electrochemical immunosensor for PCT using single-walled carbon nanohorns (SWCNHs)–hollow Pt nanospheres (HPtNPs) as signal tags. Given the high specific surface area of SWCNHs and the interesting catalytic properties of HPtNPs, the successful synthesis of SWCNHs–HPtNPs provided not only a high specific surface area for loading substantial antigen but also good catalytic properties to enhance the electrochemical signal with high sensitivity. In addition, to increase the quantity of the electrochemically active compound thionine immobilized on the surface, PAMAM, which is a class of hyper-branched polymers with a diamine core and an amido amine branching structure, was assembled on the SWCNH–HPtNP surface. A series of results demonstrated the higher sensitivity, improved stability and ideal selectivity of the novel immunosensor compared with a sensor prepared using traditional methods. Furthermore, a broad linear response from 10 pg mL−1 to 20 ng mL−1 with a detection limit of 1.74 pg mL−1 was observed under the optimized conditions of the assay. This immunosensor may provide a new option for clinical septicemia diagnosis through PCT detection.

An electrochemical strategy with molecular beacon and hemin/G-quadruplex for the detection of Clostridium perfringens DNA on screen-printed electrodes

C. perfringens is a prevalent pathogen that causes infectious diseases. It becomes viable easily but often cannot be cultured and thus escapes detection. Here, we describe an electrochemical strategy based on molecular beacon (MB), streptavidin (SA), and hemin/G-quadruplex/Fe3O4 nanocomposites. Initially, the MB forms a stable hairpin, which blocks the binding capability of the SA aptamer. After incubating with target DNA, the hairpin opens and the SA aptamer is reactivated to capture the SA/alcohol dehydrogenase (ADH)/Fe3O4 nanocomposites. Through a “sandwich” reaction, the hemin/G-quadruplex is captured on the electrode surface, and the electrochemical signal of DPV is thus obtained. Our results suggest that the use of AuNPs/graphene dramatically enlarges the surface area and enhances the immobilisation of the capture probe (MB). The combination of the Fe3O4 nanocomposite with hemin/G-quadruplex enabled the progressive amplification of the electrochemical signal. It also showed satisfying stability, reproducibility and good specificity. Compared with PCR, there were no significant differences in the recovery and regression of concentration. Thus, this SPE strategy is a promising alternative for detecting C. perfringens without bacterial culture and DNA amplification in point of care testing (POCT).

A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM) detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.