CRISPR/Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection.

BACKGROUND/PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner. METHODS: K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-ß-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation. RESULTS: Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/µL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 101 copies/µL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm2 KP could be detected. CONCLUSION: We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.

Diagnostic Efficacy of Serological Antibody Detection Tests for Hepatitis Delta Virus: A Systematic Review and Meta-Analysis.

Background and Aims Coinfection of hepatitis delta virus (HDV) with hepatitis B virus (HBV) causes the most severe form of viral hepatitis, and the global prevalence of HDV infection is underestimated. Although serological testing of anti-HDV antibodies is widely used in the diagnosis of HDV, its diagnostic efficacy remains unclear. This study aimed to evaluate the diagnostic efficacy of HDV serological tests, the results of which may assist in the diagnosis of HDV. Methods Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines were followed. The PubMed, Web of Science and Cochrane Library databases were searched from the beginning to 31 May 2023. Study quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. STATA SE was used for the meta-analysis of the sensitivity, specificity, positive likelihood ratio and negative likelihood ratio. Results Among a total of 1376 initially identified studies, only 12 articles met the final inclusion criteria. The pooled sensitivity and specificity were 1.00 (95% CI: 0.00-1.00) and 0.71 (95% CI: 0.50-0.78) for HDV total antibodies, 0.96 (95% CI: 0.83-0.99) and 0.98 (95% CI: 0.82-1.00) for anti-HDV IgM and 0.95 (95% CI: 0.86-0.98) and 0.96 (95% CI: 0.67-1.00) for anti-HDV IgG. The pooled sensitivity and specificity for HDV serological tests were 0.99 (95% CI: 0.96-1.00) and 0.90 (95% CI: 0.79-0.96). Conclusions This meta-analysis suggests that serological tests have high diagnostic performance in detecting antibodies against HDV, especially in HDV IgM and IgG. However, this conclusion is based on studies of a limited number and quality, and the development of new diagnostic tools with higher precision and reliability is still necessary.

CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection.

BACKGROUND & AIMS: Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR-Cas13a technology. METHODS: We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR-Cas13a combined with RT-PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR-Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT-qPCR and RT-ddPCR. RESULTS: For synthetic HDV RNA plasmids, the sensitivity of RT-PCR-CRISPR-based fluorescence assays was 1 copy/µL, higher than that of RT-qPCR (10 copies/µL) and RT-ddPCR (10 copies/µL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/µL, as low as that of RT-qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT-qPCR, RT-ddPCR, RT-PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively. CONCLUSIONS: We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

Performance of Hepatitis Delta Virus (HDV) RNA Testing for the Diagnosis of Active HDV Infection: Systematic Review and Meta-analysis.

Background and Aims: Hepatitis delta virus (HDV) is a defective virus and causes severe liver disease. Several HDV RNA assays have been developed, however the diagnostic efficacy remains unclear.This systematic review and meta-analysis aims to evaluate the diagnostic accuracy of HDV RNA assays to aid in the diagnosis of active hepatitis D. Methods: The PubMed, Embase, and Cochrane Library databases were systematically searched from the beginning to June 31, 2022. Information on the characteristics of the literature and data on sensitivity, specificity, and area under curve (AUC) of the receiver operating characteristic (ROC) were extracted. Stata 14.0 was used for meta-analysis of the combined sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio. Results: A total of 10 studies were included in the meta-analysis. The summary sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio of HDV RNA assays for HDV diagnosis were 0.92 (95% CI: 0.87-0.95), 0.90 (95% CI: 0.86-0.93), 7.74 (95% CI: 5.31-11.29), 0.10 (95% CI: 0.06-0.18) and 99.90 (95% CI: 47.08-211.99), respectively. The AUC of the pooled ROC curve was 0.95 (95% CI: 0.92-0.96). Conclusions: The results show that HDV RNA assays had high diagnostic performance. However, that is limited by the number and quality of studies. Standard protocols for the development of assays by manufacturers and larger studies on the use of the assays are needed.

CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients.

BACKGROUND & AIMS: The WHO declared to eliminate hepatitis B virus (HBV) by 2030. However, an increasing number of patients are presenting with low-level viremia (LLV) with the widespread use of antiviral medications. The diagnostic efficiency and coverage area of HBV infection are low. Hence, this study intended to drive the HBV infection detection to effectively adaptable for any small to medium-sized laboratory or field survey. METHODS: We established, optimized, and evaluated a colloidal gold test strip for detection of HBV DNA based on CRISPR/Cas13a combined with recombinase-aided amplification (RAA) technology. Furthermore, 180 HBV-infected patients (including patients with different viral loads, LLV patients and dynamic plasma samples of patients on antiviral therapy) were enrolled for clinical validation. RESULTS: The strip detection of HBV DNA was established based on RAA-CRISPR-Cas13a technology with a sensitivity of 101 copies/µL and a specificity of 100%. HBV DNA gradient concentration plasmids and clinical samples were effectively identified by this approach. The positive coincidence rate for LLV patients was 87%, while the negative coincidence rate was 100%. The positive coincidence rate reached 100% in LLV patients (viral loading >100 IU/mL). The sensitivity, specificity, positive predictive agreement (PPA) and negative predictive agreement (NPA) values of dynamic plasma detection in patients on antiviral therapy were 100%, 92.15%, 93.75%, and 100%, respectively. CONCLUSIONS: We develop rapid and portable RAA-CRISPR/Cas13a-based strip of HBV DNA detection for LLV patients. This study provides a visual and faster alternative to current PCR-based diagnosis for HBV infection.

Underestimated Prevalence of HIV, Hepatitis B Virus (HBV), and Hepatitis D Virus (HDV) Triple Infection Globally: Systematic Review and Meta-analysis.

BACKGROUND: Hepatitis delta virus (HDV) is a satellite RNA virus that relies on hepatitis B virus (HBV) for transmission. HIV/HBV/HDV coinfection or triple infection is common and has a worse prognosis than monoinfection. OBJECTIVE: We aimed to reveal the epidemiological characteristics of HIV/HBV/HDV triple infection in the global population. METHODS: A systematic literature search in PubMed, Embase, and the Cochrane Library was performed for studies of the prevalence of HIV/HBV/HDV triple infection published from January 1, 1990, to May 31, 2021. The Der Simonian-Laird random effects model was used to calculate the pooled prevalence. RESULTS: We included 14 studies with 11,852 participants. The pooled triple infection rate in the global population was 7.4% (877/11,852; 95% CI 0.73%-29.59%). The results of the subgroup analysis showed that the prevalence of triple infection was significantly higher in the Asian population (214/986, 21.4%; 95% CI 7.1%-35.8%), in men (212/5579, 3.8%; 95% CI 2.5%-5.2%), and in men who have sex with men (216/2734, 7.9%; 95% CI 4.3%-11.4%). In addition, compared with people living with HIV, the HIV/HBV/HDV triple infection rate was higher in people with hepatitis B. CONCLUSIONS: This meta-analysis suggests that the prevalence of HIV/HBV/HDV triple infection in the global population is underestimated, and we should focus more effort on the prevention and control of HIV/HBV/HDV triple infection. TRIAL REGISTRATION: PROSPERO CRD42021273949; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=273949.

Digital Droplet PCR for Detection and Quantitation of Hepatitis Delta Virus.

INTRODUCTION: Hepatitis delta virus (HDV) far exceeds our expected level. There remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet polymerase chain reaction (ddPCR) for HDV quantitative detection. METHODS: With plasmid (pMD19T) containing HDV full genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed with hepatitis D and 14 controls were examined using enzyme-linked immunosorbent assay, reverse-transcriptase PCR (RT-PCR), and ddPCR. A total of 728 hepatitis B virus-related patients, including 182 patients with chronic hepatitis B, 182 with liver cirrhosis, 182 with hepatocellular carcinoma, and 182 with liver failure, were screened for HDV infection. RESULTS: The detection limit of ddPCR for HDV is significantly low, with lower limit of detection and lower limit of quantitation of 0.29 IU/mL (95% confidence interval: 1.93 × 10-3-1.22 IU/mL) and 8.76 IU/mL (95% confidence interval: 1.83-1.03 × 106 IU/mL), respectively. Among the 44 samples, the enzyme-linked immunosorbent assay detected 30 cases positive, ddPCR reported 24 samples, and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV were 1.1%, 3.3%, 2.7%, and 7.1% in patients with chronic hepatitis B, liver cirrhosis, hepatocellular carcinoma, and liver failure, respectively; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4%, and 20%, respectively. However, the detection rates of ddPCR were 0%, 33.33%, 30.77%, and 60%, respectively. DISCUSSION: We establish a high sensitivity and specificity quantitative HDV RNA detection method based on ddPCR. Hepatitis B virus-related end-stage liver diseases, especially liver failure, are associated with a remarkably high rate of HDV infection.

CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection.

BACKGROUND AND AIMS: The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology. METHODS: We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs). RESULTS: Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/µl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods. CONCLUSIONS: We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.

[Corrigendum] Downregulation of heparanase by RNA interference inhibits invasion and tumorigenesis of hepatocellular cancer cells in vitro and in vivo.

Following the publication of this article, an interested reader drew to the authors' attention that two images in Fig. 1B (the a and d panels) appeared to represent the same clone, albeit with different intensities and the panels were cropped differently. The authors were able to confirm that Figs. 1B(a) and B(d) were inadvertently selected from the same set of images but with different exposure times: Owing to an error in data handling, a wrong image was chosen during the grouping the figures. The corrected version of Fig. 1 is shown on the next page, featuring the correct image for Fig. 1B(d). The authors regret that this error was not picked up upon before the paper was sent to press, although the error did not affect the major conclusions reported in the paper. The authors thank the Editor of International Journal of Oncology for allowing them the opportunity to publish a Corrigendum. and regret any inconvenience caused to the readership. [the origional article was published on International Journal of Oncology 40: 1601­1609, 2012; DOI: 10.3892/ijo.2012.1338].

[Effects of simulated acid rain on soil N2O emission from typical forest in subtropical sou-thern China.] / æ¨¡æ‹Ÿé ¸é›¨å¯¹å—äºšçƒ­å¸¦å ¸åž‹æ£®æž—åœŸå£¤N2O排放的影响.

Based on a long-term simulated acid rain experiment, soil N2O emission fluxes were measured using static chambers and the gas chromatography method in a coniferous and broadleaved mixed forest and a monsoon evergreen broadleaved forest in southern China. During the five-year observation periods (2014-2018), soil N2O emission fluxes in the two forests showed obvious seasonal variation. The soil N2O emission fluxes in wet season were significantly higher than that in dry season, with a large annual variation. Due to the decreases of precipitation, soil N2O emission fluxes of the two forests in 2017 and 2018 were generally low. Soil N2O emission flux was positively correlated with soil temperature and soil moisture. In the monsoon evergreen broadleaved forest, soil N2O emission flux in the control plot was 12.6 µg N2O·m-2·h-1. Soil N2O emission fluxes under the pH 3.5 and pH 3.0 treatments increased by 42.9% and 61.1%, respectively. Soil N2O emission was significantly increased under simulated acid rain in the monsoon evergreen broadleaved forest. Acid rain promoted soil N2O emission in the coniferous and broadleaved mixed forest, but without significant difference among the treatments. Under the scenario of increasing acid rain, soil N2O emission fluxes in typical subtropical southern China forests would increase, and the magnitude of such increase was different among forest types.

Magnesium Isoglycyrrhizinate Ameliorates Concanavalin A-Induced Liver Injury by Inhibiting Autophagy.

Background and Aims: Acute liver failure (ALF) is a type of liver injury that is caused by multiple factors and leads to severe liver dysfunction; however, current treatments for ALF are insufficient. Magnesium isoglycyrrhizinate (MgIG), a novel glycyrrhizin extracted from the traditional Chinese medicine licorice, has a significant protective effect against concanavalin A (ConA)-induced liver injury, but its underlying therapeutic mechanism is unclear. Hence, this study aims to explore the potential therapeutic mechanism of MgIG against ConA-induced immune liver injury. Methods: ConA (20 mg/kg, i. v.) was administered for 12 h to construct an immune liver injury model, and the treatment group was given MgIG (30 mg/kg, i. p.) injection 1 h in advance. Lethality, liver injury, cytokine levels, and hepatocyte death were evaluated. The level of autophagy was evaluated by electron microscopy, RT-PCR and western blotting, and hepatocyte death was assessed in vitro by flow cytometry. Results: MgIG significantly increased the survival rate of mice and ameliorated severe liver injury mediated by ConA. The decrease in the number of autophagosomes, downregulation of LC3b expression and upregulation of p62 expression indicated that MgIG significantly inhibited ConA-induced autophagy in the liver. Reactivation of autophagy by rapamycin (RAPA) reversed the protective effect of MgIG against ConA-induced liver injury. Compared with MgIG treatment, activation of autophagy by RAPA also promoted the expression of liver inflammation markers (IL-1ß, IL-6, TNF-α, CXCL-1, CXCL-2, CXCL-10, etc.) and hepatocyte death. In vitro experiments also showed that MgIG reduced ConA-induced hepatocyte death but did not decrease hepatocyte apoptosis by inhibiting autophagy. Conclusion: MgIG significantly ameliorated ConA-induced immune liver injury in mice by inhibiting autophagy. This study provides theoretical support for the ability of MgIG to protect against liver injury in clinical practice.

Use of a transjugular intrahepatic portosystemic shunt combined with autologous bone marrow cell infusion in patients with decompensated liver cirrhosis: an exploratory study.

BACKGROUND AIMS: Currently, there is no treatment for decompensated liver cirrhosis except for liver transplantation. The safety and effect on liver function of a transjugular intrahepatic portosystemic shunt (TIPS) with and without autologous bone marrow cell (BMC) infusion in patients with decompensated liver cirrhosis were determined. METHODS: Ten patients who were diagnosed with decompensated liver cirrhosis during the period from September 2011 to July 2012 were enrolled in this study. The patients underwent TIPS (TIPS group) or combined treatment with TIPS and BMC infusion through the hepatic artery (TIPS+BMC group). All patients were monitored for adverse events, liver function and complications caused by portal hypertension during a period of 52 weeks. RESULTS: The number of infused BMCs was 2.65 ± 1.20 ×10(9). Significant improvements in the serum levels of albumin and total bilirubin and decreased Child-Pugh scores were observed in patients treated with both TIPS and BMCs (P < 0.05), whereas no such changes were observed in the TIPS group. Endoscopic findings showed that varices in the esophagus and the gastric fundus were alleviated after either treatment. All 10 patients showed a complete or partial resolution of ascites at 4 weeks. No major adverse effects were noted during the follow-up period for patients in either group. CONCLUSIONS: TIPS combined with BMC infusion is clinically safe; the treatment improved liver function and alleviated complications caused by portal hypertension; therefore, this combination has potential for treatment of patients with decompensated liver cirrhosis.

Highly enhanced electrochemiluminescent strategy for tumor biomarkers detection with in situ generation of L-homocysteine for signal amplification.

In this work, an ultrasensitive peroxydisulfate electrochemiluminescence (ECL) immunosensor using in situ generation of L-homocysteine (L-Hcys) for signal amplification was successfully constructed for detection of carcinoembryonic antigen (CEA). In the reaction of biological methylation, S-adenosyl-L-homocysteine hydrolase (SAHH) catalyzed the reversible hydrolysis of S-adenosyl-L-homocysteine (SAH) to produce L-Hcys, which was inducted into ECL system to construct the immunosensor for signal amplification in this work. Simultaneously, Gold and palladium nanoparticles functionalized multi-walled carbon nanotubes (Au-PdNPs@MWCNTs) were prepared, which were introduced to immobilize the secondary antibody (Ab2) and SAHH with high loading amount and good biological activity due to their improved surface area and excellent biocompatibility. Then the proposed ECL immunosensor was developed by a sandwich-type format using Au-PdNPs@MWCNTs-SAHH-Ab2 as tracer and graphene together with AuNPs as substrate. Besides the enhancement of Au-PdNPs, the enzymatic catalysis reaction also amplified the ECL signal dramatically, which was achieved by efficient catalysis of the SAHH towards the hydrolysis of SAH to generate improved amount of L-Hcys in situ. Furthermore, due to the special interaction between Au-PdNPs and -SH or -NH2 in L-Hcys, L-Hcys would gradually accumulate on the surface of the immunosensor, which greatly enhanced the concentration of L-Hcys on the immunosensor surface and further improved the ECL intensity. With the amplification factors above, a wide linear ranged from 0.1 pg mL(-1) to 80 ng mL(-1) was acquired with a relatively low detection limit of 33 fg mL(-1) for CEA.

Amplified electrochemiluminescence of luminol based on hybridization chain reaction and in situ generate co-reactant for highly sensitive immunoassay.

In this work, we described a simple and highly sensitive electrochemiluminescence (ECL) strategy for IgG detection. Firstly, L-cysteine functionalized reduced graphene oxide composite (L-cys-rGO) was decorated on the glassy carbon electrode (GCE) surface. Then anti-IgG was immobilized on the modified electrode surface through the interaction between the carboxylic groups of the L-cys-rGO and the amine groups in anti-IgG. And then biotinylated anti-IgG (bio-anti-IgG) was assembled onto the electrode surface based on the sandwich-type immunoreactions. By the conjunction of biotin and streptavidin (SA), SA was immobilized, which in turn, combined with the biotin labeled initiator strand (S1). In the presence of two single DNA strands of glucose oxidase labeled S2 (GOD-S2) and complementary strand (S3), S1 could trigger the hybridization chain reaction (HCR) among S1, GOD-S2 and S3. Herein, due to HCR, numerous GOD was efficiently immobilizated on the sensing surface and exhibited excellent catalysis towards glucose to in situ generate amounts of hydrogen peroxide (H2O2), which acted as luminol's co-reactant to significantly enhance the ECL signal. The proposed ECL immunosensor presented predominate stability and high sensibility for determination of IgG in the range from 0.1 pg mL(-1) to 100 ng mL(-1) with a detection limit of 33 fg mL(-1) (S/N=3). Additionally, the designed ECL immunosensor exhibited a promising application for other protein detection.

An ultrasensitive electrochemiluminescence immunoassay based on supersandwich DNA structure amplification with histidine as a co-reactant.

Here we report a novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of carcinoembryonic antigen (CEA) via histidine labeled supersandwich DNA structure to amplify the ECL signal. Histidine is an α-amino acid with an imidazole group, which can act as a co-reactant of tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)3(2+)) to amplify ECL signal. To the best of our knowledge, this is the first time to use histidine as co-reactant of Ru(bpy)3(2+) for signal amplification. The ECL substrate is fabricated by locating the complex of Pt nanoparticles (PtNPs) and Ru(bpy)3(2+) (Ru-PtNPs) on Nafion modified electrode surface. In the presence of antigens, the sandwiched immunocomplex can be formed between the primary antibodies on the ECL substrate and the secondary antibodies on the gold nanoparticles. The carried auxiliary probe I trigger a cascade of hybridization events between alternating auxiliary probe I and histidine-modified auxiliary probe II to form the long DNA concatamers. Thus the long DNA concatamers contain multiple histidine-modified auxiliary probe II, which accordingly achieve significant signal amplification. As a result, the sensitivity of proposed immunosensor for CEA detection is improved, the linear range is from 0.1 pg mL(-1) to 100 ng mL(-1) with a low detection limit of 33.3 fg mL(-1). Moreover, with the satisfying stability, selectivity and reproducibility, the proposed supersandwich immunoassay performs good promise in clinical detection.

An ultrasensitive luminol cathodic electrochemiluminescence immunosensor based on glucose oxidase and nanocomposites: graphene-carbon nanotubes and gold-platinum alloy.

In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene-carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H2O2), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (-0.1 to 0.4V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL(-1) to 40 ng mL(-1) with a limit of detection down to 0.03 pg mL(-1) (SN(-1)=3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications.

Amplified cathodic electrochemiluminescence of luminol based on Pd and Pt nanoparticles and glucose oxidase decorated graphene as trace label for ultrasensitive detection of protein.

An ultrasensitive electrochemiluminescence (ECL) immunosensor was constructed for ultrasensitive detection of carcinoembryonic antigen (CEA) based on an amplified cathodic ECL of luminol at low potential. Firstly, Au nanoparticles (AuNPs) were electrodeposited onto single walled carbon nanotube-graphene composites (CNTs-Gra) coated glass carbon electrode (GCE) with enhanced surface area and good biocompatibility to capture primary antibody (Ab1) and then bind the antigen analytes. Secondly, Pd and Pt nanoparticles (Pd&PtNPs) decorated reduced graphene oxide (Pd&PtNPs@rGO) and glucose oxidase (GOD) labeled secondary antibody (Pd&PtNPs@ rGO-GOD-Ab2) could be captured onto the electrode surface by a sandwich immunoassay protocol to generate amplified cathodic ECL signals of luminol in the presence of glucose. The Pd&PtNPs@rGO composites and loaded GOD promoted luminol cathodic ECL response by efficiently catalyzing glucose to in-situ produce amount of hydrogen peroxide (H2O2) working as a coreactant of luminol. Then in turn Pd&PtNPs catalyzed H2O2 to generate various reactive oxygen species (ROSs), which accelerated the cathodic ECL reaction of luminol, enhanced the cathodic ECL intensity of luminol and improved the sensitivity of the immunosensor. The as-proposed ECL immunosensor exhibited sensitive response on the detection of CEA ranging from 0.0001 ng mL(-1) to 160 ng mL(-1) with a detection limit of 0.03 pg mL(-1) (S/N=3). Moreover, the stability, specificity, lifetime and reproducibility tests demonstrated the feasibility of the developed immunoassay, which can be further extended to the detection of other disease biomarkers.

Glucocorticoids can increase the survival rate of patients with severe viral hepatitis B: a meta-analysis.

OBJECTIVES: Severe viral hepatitis B is a disease associated with significant morbidity and mortality. Clinical controlled trials show that the efficacy of treatment of severe viral hepatitis B with glucocorticoids remains debatable. Therefore, we carried out this meta-analysis to evaluate the safety, efficacy, and side effects of glucocorticoid therapy for severe viral hepatitis B. METHODS: We searched PubMed, Medline, Embase, Cochrane Library, and Google Scholar for randomized-controlled trials published before April 2012 in which glucocorticoid therapy was compared with routine treatment for severe viral hepatitis B. The primary outcome was the survival rate of the two groups. RESULTS: We selected eight controlled clinical trials, which included 597 patients. We recorded a benefit of glucocorticoid treatment on the survival rate of patients with severe viral hepatitis B (597 patients) [risk ratio (RR)=1.188, 95% confidence interval (CI) 1.030-1.369, P=0.018]. The benefit was most noticeable in patients at the stage of preliver failure (409 patients) (RR=1.275, 95% CI 1.077-1.510, P=0.005), whereas there was no efficacy for patients with liver failure (188 patients) (RR=1.008, 95% CI 0.774-1.312, P=0.955). Glucocorticoid treatment was not associated with the development of secondary infection and bleeding. CONCLUSION: Treatment with glucocorticoids can significantly increase the survival rate of patients with severe hepatitis B. The benefit was most noticeable in patients at the stage of preliver failure. However, the incidence of secondary infection and bleeding did not change significantly. This finding suggests that prompt and timely glucocorticoid treatment is crucial.

An ultrasensitive peroxydisulfate electrochemiluminescence immunosensor for Streptococcus suis serotype 2 based on L-cysteine combined with mimicking bi-enzyme synergetic catalysis to in situ generate coreactant.

A novel signal amplification strategy of mimicking bi-enzyme synergetic catalysis to generate coreactant in situ was designed to fabricate an ultrasensitive peroxydisulfate electrochemiluminescence (ECL) immunosensor for detection of Streptococcus suis serotype 2 (SS2). It was the first time to detect SS2 by using ECL. Through the interaction between l-cysteine (l-cys) and hollow PtPd bimetal alloy nanoparticles (HPtPd) to form ((l-cys-HPtPd)n) nanocomposites, the loading amount of l-cys and HPtPd was greatly increased, which could greatly enhance the ECL signal of peroxydisulfate. At the same time, Glucose Oxidase (GOD), used to block nonspecific binding sites of (l-cys-HPtPd)n nanocomposites, could rapidly oxidize d-glucose in the detection solution into gluconic acid accompanying with the generation of H2O2, which was further catalyzed by HPtPd to generate O2. And O2, acted as the coreactant of peroxydisulfate, could greatly amplify the ECL signal. In the process, HPtPd could be regarded as mimicking enzyme, the effect of which was similar to horseradish peroxidase (HRP) in generating O2. With the several amplification factors of a sandwich-type structure we designed, a wide linear ranged from 0.0001 to 100ngmL(-1) was acquired with a relatively low detection limit of 33fgmL(-1) for SS2. The present work demonstrated that the novel strategy had the great advantages in sensitivity, selectivity and reproducibility which might hold a new promise for highly sensitive bioassays applied in clinical detection.

Highly amplified electrochemiluminescence of peroxydisulfate using bienzyme functionalized palladium nanoparticles as labels for ultrasensitive immunoassay.

An immunosensor based on the electrochemiluminescence (ECL) of peroxydisulfate was firstly proposed by coupling the cooperation of two enzymes to in situ generate coreactant with palladium nanoparticles (PdNPs) as catalyst for the ECL reaction. PdNPs were previously synthesized, which successfully attached to functional carbon nanotubes (FCNTs), to bind the secondary antibody and bienzyme (horseradish peroxidase and glucose oxidase). Then the prepared bioconjugates were introduced to the electrode via sandwich immunoreactions. Accordingly, a dramatically amplified ECL signal was obtained for that GOD catalyzed glucose to produce H(2)O(2) which was subsequently reduced by HRP to in situ generate O(2), then PdNPs as catalyst for the ECL reaction of peroxydisulfate/O(2). The present immunosensor was used to detect α-1-fetoprotein (AFP) and showed a wide linear range of 1×10(-5)-100ng mL(-1), with a low detection limit of 3.3fg mL(-1)(S/N=3). This new signal amplification strategy for preparation of the ECL immunosensor could be easily realized and has a potential application in ultrasensitive bioassays.