Co-reporter:Ruihua Fei, Tongtong Zhang, Yue Huang, Yonggang Hu
Analytica Chimica Acta 2017 Volume 986(Volume 986) pp:
Publication Date(Web):15 September 2017
DOI:10.1016/j.aca.2017.07.030
•A novel biomaterial, spore@Fe3+ microsphere, was developed for selective enrichment of phosphoproteins.•High adsorption capacity for phosphoproteins (1983 and 1818 mg g−1 for α-casein and β-casein, respectively).•High selectivity in the mixtures of phosphoprotein and non-phosphoprotein, and in real samples.•Spore@Fe3+ microsphere presents several advantages, such as easy synthesis, low cost, and ecological friendliness.The advantages of spore-based microspheres include high monodispersity, presence of different functional groups (carboxylic, amino, and hydroxyl groups), facile industrial-scale preparation by using cell cultures via fermentation in a potentially cost effective and environment friendly manner, and high uniformity. We developed a novel spore@Fe3+ microsphere for specific capture of phosphoproteins. Caseins (α-casein and β-casein) as phosphoproteins were used to evaluate binding capacity and enrichment factor. The spore@Fe3+ microspheres demonstrate high binding capacity and selectivity for phosphoproteins (1983 and 1818 mg g−1 for α-casein and β-casein, respectively). A mixture of bovine serum albumin and β-casein at 100:1 ratio displayed an enrichment factor higher than 173-fold, which can nearly be considered “purification” of phosphoproteins. The proposed method is a promising technique in developing more selective, rapid, low cost, and high-throughput platforms for phosphoprotein enrichment, and it presents potential application in investigation of protein functions and in personalized diagnostic tests.Download high-res image (208KB)Download full-size image
Co-reporter:Tian Wang, Yuqiang Xiang, Xiaoxiao Liu, Wenli Chen, Yonggang Hu
Talanta 2017 Volume 162() pp:143-150
Publication Date(Web):1 January 2017
DOI:10.1016/j.talanta.2016.10.006
•The fluorescence method for laccase assay based on Amplex Red was demonstrated.•The mechanism relies on dissolved oxygen.•The method was used to detect laccase activities in real samples.In this paper, a novel fluorescence-based method for laccase assay was presented. The method was based on the transformation of Amplex Red into a highly fluorescent and colored resorufin catalyzed by laccase in the presence of O2. The catalysis and transformation mechanism were investigated in detail. The kinetic parameters of the Amplex Red catalysis by laccase were determined using the Lineweaver–Burk equation. Vmax and Km were estimated to be 15.63 μmol min−1 and 76.88 μmol L−1, respectively. Under optimal conditions, a good linear correlation was found between fluorescence intensity and laccase activities within 5.62–702 U L−1 (r=0.9992), with a detection limit of 1.76 U L−1 (S/N=3). A series of repeatability measurements (351 U L−1 laccase) gave reproducible results with a relative standard deviation (RSD) of 1.9% (n=11). The recoveries ranged from 93.7% to 100.0% after standard additions. Common existing species such as Mg2+, Zn2+, Ni2+, Al3+, Co2+, Cd2+, K+, Ca2+, Na+, Fe3+, Li+, Cu2+, Mn2+, Fe2+, l-lysine, glycine, glucose, phenol, humic acid, lignin peroxidase, manganese peroxidase alkaline phosphatase, cellulose, glucose oxidase, urease, catalase, invertase, and horseradish peroxidase did not significantly exhibit interference. The test solution (i.e., Amplex Red stock solution) could stabilize at least three months via storage in dark at 4±0.1 °C. These results confirmed that the laccase–Amplex Red system was stable and reproducible with strong anti-interference ability and good selectivity, suggesting that this method can has great potential in practical applications for the assay of laccase activity. The proposed method was further successfully used to detect laccase activities in 38 soil samples. We noticed that the laccase activity significantly correlated with total nitrogen content (r=0.559; p<0.01) of soil, indicating laccase activity assay holds great promise as an index of soil analysis. These findings indicate that this presented method has great perspective in ecological investigation and fundamental research of soil environment.
Co-reporter:Zhiming Zeng, Yin Zhong, Huicui Yang, Ruihua Fei, Rui Zhou, Rafael Luque and Yonggang Hu
Green Chemistry 2016 vol. 18(Issue 1) pp:186-196
Publication Date(Web):20 Aug 2015
DOI:10.1039/C5GC00630A
A novel class of bionanocomposites based on monodisperse microparticles containing metal nanoparticles including Au, Pd, Ag and Pt were synthesized and characterized using a simple and efficient approach. The versatile nanocomposites exhibited unique possibilities as new biosensor for highly selective and sensitive immunoassays as well as a remarkable enhancement in catalytic activity in the reduction of 4-nitrophenol selected as model reaction which illustrated their potential in various different applications.
Co-reporter:Xingya Zhang, Zheng Li, Tao Zhou, Qian Zhou, Zhiming Zeng, Xiangdong Xu, Yonggang Hu
Talanta 2016 Volume 150() pp:184-189
Publication Date(Web):1 April 2016
DOI:10.1016/j.talanta.2015.12.029
•QD@Spore nanocomposites are synthesized as a pH sensor.•These nanocomposites are sensitive to pH in a broad range of 5.0–10.0.•The possible mechanism and stability of nanocomposites are investigated in detail.•The developed sensors have been applied for pH estimation of real samples.A new quantum dot (QD)-based pH sensor design is investigated. The sensor is synthesized based on the self-assembly of green QDs onto treated spores to form QD@spore nanocomposites. The nanocomposites are characterized using laser scanning confocal microscopy, transmission electron microscope, and fluorescence spectroscopy, among others. Fluorescence measurements showed that these nanocomposites are sensitive to pH in a broad pH range of 5.0–10.0. The developed pH sensors have been satisfactorily applied for pH estimation of real samples and are comparable with those of the commercial assay method, indicating the potential practical application of the pH sensors.
Co-reporter:Zhiming Zeng, Longjian Tian, Zheng Li, Lina Jia, Xinya Zhang, Miaomiao Xia, Yonggang Hu
Biosensors and Bioelectronics 2015 Volume 69() pp:162-166
Publication Date(Web):15 July 2015
DOI:10.1016/j.bios.2015.02.032
•A whole-cell method for phenol spectrophotometric determination was presented.•This technique was based on the color reaction catalyzed by B. amyloliquefaciens endospores.•The catalytic activity of the endospores was attributed to the presence of coat protein A.A green method for phenol spectrophotometric determination was developed based on the color reaction of phenol with 4-aminoantipyrine catalyzed by addition of Bacillus amyloliquefaciens endospores in the presence of O2. The catalytic activity of the endospores may be attributed to the presence of coat protein A on the cell surfaces. This deduction was confirmed by cotA gene knock-out from B. amyloliquefaciens using the homologous double-exchange method. Under optimal conditions, linear responses were obtained over phenol concentrations ranging from 5.0×10−5 g L−1 to 1.0×10−2 g L−1 (r=0.9984) with a detection limit of 2.1×10−5 g L−1 (3σ). Repeatability measurements of 1.0 mg L−1 phenol provided reproducible results with a relative standard deviation of 5.3% (n=11). Standard addition tests indicated recoveries ranging from 92.78% to 107.60%. The proposed whole-cell method was successfully used to detect total phenol in synthetic samples. Results confirmed the potential use of the developed method in practical applications.
Co-reporter:Xi Yang;Tao Zhou;Lei Yu;Wenwen Tan;Rui Zhou;Yonggang Hu
Luminescence 2015 Volume 30( Issue 2) pp:228-234
Publication Date(Web):
DOI:10.1002/bio.2718
ABSTRACT
A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05–80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2. Copyright © 2014 John Wiley & Sons, Ltd.
Co-reporter:Yun Zhang, Chen Tan, Ruihua Fei, Xiaoxiao Liu, Yuan Zhou, Jing Chen, Huanchun Chen, Rui Zhou, and Yonggang Hu
Analytical Chemistry 2014 Volume 86(Issue 2) pp:1115
Publication Date(Web):December 26, 2013
DOI:10.1021/ac4028774
A novel, sensitive chemiluminescence (CL) immunoassay for Escherichia coli O157:H7 detection with signal dual-amplification using glucose oxidase (GOx) and laccase was investigated. The method was based on the characterization of a luminol–H2O2–laccase reaction. Compared with the horseradish peroxidase-based biosensor, laccase exhibited high catalytic activity in strong alkaline medium, which was compatible with the luminol system. The capture antibody was immobilized onto the magnetic bead (MB) surfaces. The detection antibody was linked with GOx through biotin–avidin recognition. Accordingly, the bioconjugation of MB–caputure antibody–E. coli O157:H7–detection antibody–GOx catalyzed the substrate glucose, thereby generating H2O2. E. coli O157:H7 was then detected by measuring the CL intensity after H2O2 formation. Under optimal conditions, the calibration plot obtained for E. coli O157:H7 was approximately linear from 4.3 × 103 colony-forming unit (CFU) mL–1 to 4.3 × 105 CFU mL–1, and the total assay time was <2.0 h without any enrichment. The limit of detection for the assay was 1.2 × 103 CFU mL–1 (3σ), which was considerably lower than that of enzyme-linked immunosorbent assay method (1.0 × 105 CFU mL–1) (3σ). A series of repeatability measurements of using 1.7 × 104 CFU mL–1 E. coli O157:H7 exhibited reproducible results with a relative standard deviation (RSD) of 3.5% (n = 11). Moreover, the proposed method was successfully used to detect E. coli O157:H7 in synthetic samples (spring water, apple juice, and skim milk), which indicated its potential practical application. This protocol can be applied in various fields of study.
Co-reporter:Lina Jia, Ruihua Fei, Xinya Zhang, Haixia Tang, and Yonggang Hu
Analytical Chemistry 2014 Volume 86(Issue 23) pp:11578
Publication Date(Web):November 12, 2014
DOI:10.1021/ac500866r
A novel endospore-based microbial method for “post-additional” antioxidant capacity assay was developed. The technique was based on oxidation and catalysis of the 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) by Bacillus subtilis 168 endospores in the presence of dissolved oxygen. Coat protein A (CotA), which belongs to the endospore coat, was expressed, purified, and assessed for its ability to oxidize ABTS into the ABTS•+ radical cation. The wild-type endospore necessary for oxidizing ABTS into ABTS•+ radical cation was confirmed by knocking out the cotA gene from B. subtilis 168 by homologous double exchange. Findings revealed that the catalytic activity of the endospores may be attributed to the presence of the CotA protein. The use of endospores instead of purified enzymes to prepare ABTS•+ greatly reduced the assay cost and eliminated the need to purify and store of enzymes. The self-life of the radical cation was kept stable for at least 12 days without addition of a stabilizer and laccase inhibitor. This behavior enables the large-scale preparation of ABTS•+. The antioxidant capacities of the individual antioxidants and fruit samples were easily quantified and compared using the proposed method. The developed technique can be further developed as a high-throughput screening technique for antioxidants.
Co-reporter:Yuan Zhou;Tao Zhou;Rui Zhou;Yonggang Hu
Luminescence 2014 Volume 29( Issue 4) pp:338-343
Publication Date(Web):
DOI:10.1002/bio.2549
ABSTRACT
A rapid, simple, facile, sensitive and enzyme-amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co-immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle-based nanoprobes. This nanoprobe was employed in a sandwich-type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP–luminol with H2O2. Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme-linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme-linked immunosorbent assay results. Copyright © 2013 John Wiley & Sons, Ltd.
Co-reporter:Huicui Yang, Xiaoxiao Liu, Ruihua Fei, Yonggang Hu
Talanta 2013 Volume 116() pp:548-553
Publication Date(Web):15 November 2013
DOI:10.1016/j.talanta.2013.07.041
•The Ag+ ions were selectively deposited on the surfaces of Fe3O4@Au nanoparticles.•A simple and selective magnetic electrochemical method for the detection of Ag+ ions in aqueous solutions was developed.•This concept offers a new platform for the selective capture and detection of analytes.Owing to the selective deposition reaction on the surface of magnetic nanoparticles, we reported a simple and selective magnetic electrochemical method for the detection of Ag+ ions in aqueous solutions. The analyte deposited on the nanoparticles was brought to the surface of a homemade magnetic electrode and detected electrochemically in 0.1 mol/L KCl solution based on the reaction of Ag0 transferred to AgCl. Under the optimal conditions, the linear response range of Ag+ ions was 0.117–17.7 μmol/L (R2=0.9909) with a detection limit of 59 nmol/L (S/N=3). A series of repeatability measurements 1.0 μmol/L Ag+ gave reproducible results with a relative standard deviation (RSD) of 4.5% (n=11). The interference from other metal cations can be eliminated by adding EDTA as a co-additive to mask the metal cations. The recoveries ranging from 98.6% to 103.99% after standard additions demonstrate that this sensor has great potential in practical applications. The advantages of this developed method include remarkable simplicity, low cost, and no requirement for probe preparation, among others.
Co-reporter:Bo Wei, Fang Li, Huicui Yang, Lei Yu, Kaihong Zhao, Rui Zhou, Yonggang Hu
Biosensors and Bioelectronics 2012 Volume 35(Issue 1) pp:390-393
Publication Date(Web):15 May 2012
DOI:10.1016/j.bios.2012.03.027
In this paper, a simple, easily-operated and enzyme-amplified fluorescence immunoassay method using magnetic particles for the detection of antibody against Actinobacillus pleuropneumoniae (APP) has been presented. The A protein of APP Repeats-in-Toxin IV (ApxIVA) with high specificity to the APP species was immobilized onto the magnetic bead surfaces. Horseradish peroxidase (HRP), which can catalyze the substrate 4-hydroxyphenylacetic acid (p-HPA), generating fluorescent bi-p, p′-hydroxyphenylacetic acid (DBDA), was selected as an enzymatic-amplified tracer. The ApxIVA antibody was detected for the presence of APP infection by measuring the fluorescence intensity of DBDA. Under optimal conditions, the calibration plot obtained for standard positive serum was approximately linear within the dilution range 1:160–1:5120. The limit of detection (LOD) for the assay was 1:10240, considerably lower than that of ApxIVA-ELISA (1:320) (S/N = 3). A series of repeatability measurements of using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of 4.8% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of 89.7%, sensitivity of 90.9% and specificity of 89.3% compared with ApxIVA-ELISA.Highlights► A MBs-based immunoassay method was developed for diagnosis of APP infection. ► This proposed method was simple, easily-operated and inexpensive. ► This immunosensor has been successfully used for the analysis of clinical samples.
Co-reporter:Fang Li, Li Mei, Yaoming Li, Kaihong Zhao, Huanchun Chen, Peng Wu, Yonggang Hu, Shengbo Cao
Biosensors and Bioelectronics 2011 Volume 26(Issue 10) pp:4253-4256
Publication Date(Web):15 June 2011
DOI:10.1016/j.bios.2011.04.028
A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2 × 103 and 5 × 105 PFU/mL. The detection limit for JEV was 2.0 × 103 PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.
Co-reporter:Fang Li, Rui Zhou, Kaihong Zhao, Huanchun Chen, Yonggang Hu
Talanta 2011 Volume 87() pp:302-306
Publication Date(Web):15 December 2011
DOI:10.1016/j.talanta.2011.09.049
A novel magnetic electrochemical immunosensor has been developed for the detection of pseudorabies virus antibody in swine serum. The magnetic glass carbon electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and gold nanoparticles were chosen as electroactive labels for the electrochemical detection. The parameters concerning the assay strategy were carefully investigated. Under the optimal conditions, the linear response range of pseudorabies virus antibody dilution ratio (standard positive serum) was 1:250 to 1:1000 with a detection limit of 1:1000. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.Highlights► A novel magnetic glass carbon electrode (MGCE) was developed to capture magnetic beads for the direct sensing application. ► The MGCE was inexpensive, robust, user-friendly, and had a commercial promise for electrochemical detection. ► A novel magnetoimmunosensor was developed, and it was successfully applied in detection of pseudorabies virus antibody.
Co-reporter:Hui Jing Zhang, Yong Gang Hu, Yong Qian Wang, Jie Zhang
Chinese Chemical Letters 2010 Volume 21(Issue 8) pp:951-954
Publication Date(Web):August 2010
DOI:10.1016/j.cclet.2010.02.025
The possibility of direct analytical applications of ferrate (VI) solution, which was freshly electrogenerated in low-concentration NaOH electrolyte, was studied by a flow-injection–chemiluminescence (FI–CL) system. It was found that some inorganic ions, organic molecule and biomolecule could enhance the chemiluminescence emission caused by ferrate (VI)–luminol reaction. V(V), Ca(II), Mg(II), phloroglucinol, and bovine hemoglobin (Hb) chosen as samples were successfully detected by this developed method. The analytical characteristics of the system for the analytes determination including linear ranges, correlation coefficients, limits of detection combined with FI analysis were studied.
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