-
[show abstract]
[hide abstract]
ABSTRACT: In this paper, we report a "signal-on" electrochemical aptasensor for simultaneous determination of two tumor markers MUC1 and VEGF(165), by using a ferrocene-labeled aptamer-complementary DNA (cDNA) as probe. Since the cDNA immobilized on an electrode surface can hybridize with both MUC1 aptamer and VEGF(165) aptamer to form a long double strand with ferrocene far away from the electrode surface, the probe cannot give electrochemical signal. Nevertheless, the presence of the two tumor markers will inhibit the hybridization of cDNA with the aptamers, thus the distance between ferrocene and the electrode is changed, and a "signal-on" electrochemical method to detect two tumor markers is developed. Experimental results show that the electrochemical signal increases with the addition of either tumor markers, but the biggest electrochemical signal can only be obtained when both tumor markers are present. Therefore, the proposed electrochemical aptasensor can not only detect the two markers but also distinguish their co-existence. It may also display high selectivity and sensitivity towards the detection of the tumor markers, so it might have potential clinical application in the future.
Biosensors & bioelectronics 02/2012; 34(1):249-52. · 5.43 Impact Factor
-
Xinjian Liu,
Hong Lin,
Qi Tang,
Chen Li,
Songtao Yang,
Zhongcan Wang,
Changjun Wang,
Qing He,
Brian Cao,
Zhenqing Feng,
Xiaohong Guan,
Jin Zhu
[show abstract]
[hide abstract]
ABSTRACT: Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×10(8), was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP).
PLoS ONE 01/2011; 6(5):e19848. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. It is necessary to develop standard rabies virus diagnostic tools, especially for diagnosing the strains prevalent in China.
Monoclonal antibodies (MAbs) specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay (ELISA), isotyping, affinity assay, immunofluorescence assay (IFA), and immunocytochemistry. The MAb, whose affinity was higher for antigen, was used to establish an antigen capture-ELISA (AC-ELISA) detection system and test the efficiency by using clinical samples.
The heavy chain subclasses of two MAbs were all determined to be IgG2a. The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis, whereas the 3C7 MAb showed the highest affinity for antigen. IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples. Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.
It was potentially useful for the further development of highly sensitive, easily handled, and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic, especially in China.
Journal of biomedical research. 09/2010; 24(5):395-403.
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factors receptor 2 (VEGFR-2) has been implicated in playing an important role in the formation of new blood vessels in tumors and other diseases. A high affinity human/mouse cross-reactive anti-VEGFR-2 monoclonal antibody (mAb) named A8H1 was established by hybridoma technology. Several immunological methods were used to characterize the A8H1, including ELISA, affinity and kinetics assay, MALDI-TOF MS, WB, IP, IF, FASC and IHC. The results suggested that A8H1 could bind with linear and conformational epitopes of the VEGFR-2 antigen. The mAb had good specific reactivity with three forms of VEGFR-2 in HUVEC, and two forms in NIH-3T3 mouse fibroblast cells, which are regarded as non-expressive for VEGFR-2. The A8H1 mAb associated with intracellular and plasma membranes in HUVEC and with the nuclei in NIH-3T3 cells. This mAb also effectively identified VEGFR-2 over-expressing cells in a number of archived human cancer tissues.
Cytotechnology 04/2010; 62(1):61-71. · 1.21 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have developed a simple electrochemical method to detect DNA damage caused by the photovoltaic effect of nano-TiO(2). Meanwhile, we have found that resveratrol, a Chinese Traditional Medicinal Herb species, can have a repairing effect to the oxidized DNA, which can also be detected with the proposed technique in this paper.
Journal of Biotechnology 02/2007; 127(4):653-6. · 3.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Horseradish peroxidase (HRP) was incorporated in dipalmitoylphosphatidic acid (DPPA) to form a film and the film was modified on pyrolytic graphite electrode. UV-Vis spectra suggested that HRP in the film could keep its secondary structure similar to the native state. A pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks was observed with the formal potential at -276.2 mV (vs. saturated calomel electrode), characteristic of heme Fe(III)/Fe(II) redox couple of HRP. The apparent heterogeneous electron transfer rate constant and other electrochemical parameters were presented. The catalytic activity of HRP in DPPA film toward oxygen, hydrogen peroxide and nitric oxide were also examined.
Bioelectrochemistry 02/2006; 68(1):98-104. · 3.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hemoglobin (Hb) can take direct electron-transfer reactions after being entrapped in poly-3-hydroxybutyrate (PHB) film. A pair of well-defined, quasi-reversible cyclic voltammetric peaks is thus obtained at an Hb-PHB modified pyrolytic graphite electrode. The anodic and cathodic peaks are located at -224 and -284 mV for a pH 5.0 acetate buffer solution. Meanwhile, the peroxidase activity of the protein in the membrane has been greatly enhanced, with the apparent Michaelis-Menten constant calculated to be 1076 microM. According to the direct electron transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb-PHB based hydrogen peroxide biosensor is prepared, with a linear range 6.0 x 10(-7) to 8.0 x 10(-4) M. The pathway of reductive dehalogenation of trichloroacetic acid is also discussed in detail. The highly reduced form of Hb produced in PHB film can be used to dechlorinate di- and monochloroacetic acid. The catalytic ability of Hb toward the reduction of nitric oxide has been investigated as well. Due to its biodegradability, low cost, chemical inertness, and especially its biocompatibility and non-toxicity, PHB would be a desirable film in the sensor field.
Biosensors and Bioelectronics 04/2005; 20(9):1836-42. · 5.60 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hemoglobin (Hb) was successfully immobilized in dimethyldioctadecyl ammonium bromide (DOAB) film at pyrolytic graphite (PG) electrode. Electrochemical experiments revealed that Hb in DOAB film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.160 V versus saturated calomel electrode (SCE) in pH 5.0 buffer, characteristic of the heme Fe(III)/Fe(II) redox couple of Hb. The electron transfer (eT) rate between Hb and the PG electrode was 0.10 s(-1). Positions of the Soret absorbance band indicated that the Hb retained its secondary structure and was similar to its native state. Furthermore, the Hb in DOAB film acted as a biological catalyst towards the reduction of nitric oxide (NO). The voltammetric response of NO at the Hb-DOAB modified electrode could be used to determine the concentration of NO in solution.
Journal of Biochemical and Biophysical Methods 03/2005; 62(2):143-51. · 2.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, we investigated redox thermodynamics of myoglobin as well as the ionic (phosphate ions) and ligation (imidazole) effects via a dynamic electrochemical approach. We employed a previously established system that features nonmediated, direct electrochemistry of myoglobin and myoglobin in an immobilized state (i.e., diffusionless electrochemistry). Thermodynamics parameters were obtained by measuring redox potential (E degrees ') of myoglobin at varied temperature (T), in the presence and in the absence of specific ions or axial ligands. As a step further, we evaluated contributions from allosteric effect and axial iron ligation by partitioning E degrees ' changes into entropic and enthalpic terms. Compensation phenomena between the entropic and enthalpic changes were observed in all these cases. On the basis of these studies, we also correlated these phenomena to possible structural variations.
Langmuir 02/2005; 21(1):375-8. · 4.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Silver nanoparticles (11+/-1.5 nm) could greatly enhance the electron-transfer reactivity of myoglobin (Mb) and its catalytic ability toward hydrogen peroxide (H2O2). Direct fast electron transfer between Mb and a pyrolytic graphite (PG) electrode was achieved, and a pair of well-defined, quasi-reversible redox peaks was obtained. The cathodic and anodic peaks were located at -329 and -281 mV, respectively. Meanwhile, the catalytic ability of the protein toward the reduction of H2O2 was also studied, and a H2O2 biosensor was subsequently fabricated. Its detection limit was 1.0 x 10(-6) M with a sensitivity of 0.0205 microA per microM of H2O2. The apparent Michaelis-Menten constant was calculated to be 1303 muM. Flocculation assay showed that the protein maintained plasmon layers surrounding the surface of silver nanoparticles and avoided silver-nanoparticle aggregation. On the other hand, UV-visible spectroscopy studies revealed that silver nanoparticles could induce a small change of the heme-group environment of the protein; this contributed to the enhancement of the electron-transfer reactivity and the catalytic activity.
ChemBioChem 01/2005; 5(12):1686-91. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel and stable film made from polyethylene glycol (PEG) on pyrolytic graphite (PG) electrode was presented in this paper for incorporating horseradish peroxidase (HRP) to study the direct electrochemistry of the enzyme. In PEG film, HRP showed a thin-layer electrochemistry behavior. The apparent standard potential (E degrees ') was -0.379 V versus SCE at pH 7.2. Moreover, the PEG-HRP modified electrode exhibited excellent electrocatalytical response to the reduction of H2O2 with a calibration range between 2.0 x 10(-6) and 6.0 x 10(-4) M and a good linear relation from 2.0 x 10(-6) to 1.0 x 10(-4) M, on which an unmediated H2O2 biosensor was based. The detection limit of 6.7 x 10(-7) M was estimated when the signal-to-noise ratio was 3. The relative standard deviation (R.S.D.) was 4.7% for six successive determinations at a concentration of 4.0 x 10(-5) M. The apparent Michaelis-Menten constant (Km app) of the sensor was found to be 1.38 mM. Epinephrine, dopamine, and ascorbic acid did not interfere with the sensitive determination of H2O2.
Biosensors and Bioelectronics 11/2004; 20(3):533-7. · 5.60 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Direct electron transfer between hemoglobin and gold electrode is achieved at both a bare and a 4, 4'-bipyridine-modified gold electrode in the presence of chloride ions. The addition of chloride to hemoglobin solution also increases the reversibility of the direct electrochemistry and shifts the formal potential of hemoglobin to the negative direction. While the existence of chloride does not significantly change the tertiary structure of the protein, it might induce a slight variation of the structure, which is beneficial to the electrochemical response. It is suggested that the chloride binding to hemoglobin is a combination of specific and unspecific bindings.
Bioelectrochemistry 09/2004; 64(1):23-7. · 3.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Direct electrochemistry of horseradish peroxidase (HRP) embedded in film of phosphatidylcholine (PC) is investigated at a pyrolytic graphite electrode by voltammetric methods. The electron-transfer reactivity between incorporated HRP and the electrode is found to be greatly enhanced by phosphatidylcholine film. Cyclic voltammetry (CV) of this incorporated peroxidase shows a pair of well-defined and nearly reversible peaks, and the cathodic and anodic peak potentials are located at about -0.261 and -0.180 V, respectively versus saturated calomel electrode at pH 5.5. Ultraviolet-visible absorption spectra indicate that the heme microenvironment of HRP in phosphatidylcholine film is similar to that of its native status. It is also observed that HRP modified electrode is able to catalyze the electrochemical reduction of nitric oxide. Experimental results reveal that the peak current related to nitric oxide reduction is linearly proportional to its concentration in the ranges of 2.0 x 10(-7) -5.0 x 10(-6) mol (-1) and 2.0 x 10(-5) -1.0 x 10(-4) mol(-1), based on which an unmediated biosensor for nitric oxide is developed.
Journal of Biotechnology 04/2004; 108(2):145-52. · 3.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Direct electron transfer of hemoglobin (Hb) can be achieved in a Hb/montmorillonite (MMT)/polyvinyl alcohol multi-assembly at a pyrolytic graphite (PG) electrode. Accordingly, a novel nitric oxide (NO) biosensor is proposed. The reduction of NO is observed at a potential of approximately -783 mV (vs. SCE) at pH 5.5. At optimum pH, this biosensor shows a wide linear range of 1.0x10(-6)-2.5x10(-4) mol/l with a detection limit of 5.0x10(-7) mol/l. The sensor-to-sensor reproducibility is good consideringmacr; a relative standard deviation of 3.5% in five independent determinations at 5.0x10(-5) mol/l NO. The modified electrode is conveniently constructed and durable in long-term operations.
Biosensors and Bioelectronics 01/2004; 19(5):441-5. · 5.60 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The titratable potentiometric response of hemoglobin (Hb) induced by adenosine-5'-triphosphate (ATP) is observed. The concentration-dependent effect of ATP on the anaerobic redox reaction of the protein at pH 7.0 reflects that ATP will induce stabilization of the reduced state and destabilization of the R-like (met Fe(III)) state of the metHb, when ATP concentration is lower than 3.0 mM. But when ATP concentration is between 4 and 7 mM, shift of the oxidation potential may also be observed. With reference to the study of adenosine, adenosine-5'-monophosphate, adenosine-5'-diphosphate and 2,3-diphosphoglycerate, the allosteric effect of ATP on Hb is discussed extensively. This study has given an electrochemical approach to the investigation of effect of ATP, an in vivo allosteric effector, on Hb in the physiological concentration range.
Biophysical Chemistry 01/2004; 106(3):267-73. · 2.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An unmediated NO biosensor was prepared by co-immobilizing haemoglobin with polyethyleneimine at a pyrolytic graphite electrode surface. Haemoglobin exhibits direct electrochemistry in the membrane, and this modified electrode shows high stability and catalytic activity towards the reduction of NO. A linear relationship between the catalytic current and the concentration of NO was observed in the concentration ranges 1.0 x 10(-7)-8.0 x 10(-6) M and 1.0 x 10(-5)-2.5 x 10(-5) M, with a detection limit of 4.0 x 10(-8) M.
Biotechnology and Applied Biochemistry 11/2003; 38(Pt 2):119-22. · 1.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A reagentless nitric oxide (NO) biosensor was prepared using a pyrolytic graphite (PG) electrode modified with a composite film containing horseradish peroxidase (HRP) and kieselguhr. Noticeably, the electron-transfer reactivity of HRP was significantly enhanced when incorporated in the inorganic kieselguhr material. Consequently, we observed the direct electrochemical response of HRP in this composite film, which would be otherwise electrochemically silent (in the absence of kieselguhr). Importantly, this modified electrode demonstrated nice catalytic activity, as well as high stability, towards the reduction of NO. The peak current related to NO was linearly proportional to its concentration from 2.0 x 10(-7) to 2.0 x 10(-5) mol/L, and the relative standard deviation was 4 % for five successive determinations at a NO concentration of 1.0 x 10(-5) mol/L. The critical level in concentration was estimated to be (4.0 +/- 0.3) x 10(-8) mol/L.
Annali di Chimica 94(5-6):457-62. · 0.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Here we report that human telomere guanine-rich sequence AGGG(TTAGGG)3 (G4) and hemin can form a G-quadruplex–hemin complex (GHC) which exhibits good electrochemical characteristics. The redox signals of the complex and the electrocatalytic activities toward the reduction of hydrogen peroxide have been investigated. Based on this, two small molecular ligands of G-quadruplex, also known as candidates of anticancer drug, 5,10,15,20-tetra-(N-methyl-4-pyridyl) porphyrin (TMPyP4) and N,N′-bis[2-(1-piperidino)-ethyl]-3,4,9,10-perylenetetracarboxylic diimide (PIPER), were used as model molecules to interrogate their binding activities and their effects on electrochemical behavior of GHC. Besides, spectrometric methods were simultaneously used to verify the interactions and binding stoichiometry between these molecules and GHC. These studies may provide a new way to characterize the structure and properties of human telomere and may have further applications.
Electrochimica Acta.
-
[show abstract]
[hide abstract]
ABSTRACT: A new film of Triton X-100 to incorporate hemoglobin for biosensor fabrication is presented in this paper. In this film, hemoglobin displays a pair of redox peaks in pH 6.0 NaAc–HAc buffer solution with a formal potential of −0.257 V (versus SCE) and shows a thin-layer behavior. Without electron mediator or promoter, the modified electrode has an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2), which shows a linear dependence on the H2O2 concentration ranging from 1.0 × 10−6 to 1.0 × 10−4 mol/L. The detection limit of 3.0 × 10−7 mol/L is estimated when the signal-to-noise ratio is 3. The value of hemoglobin in Triton X-100 film has been determined to be 4.27 mmol/L. Ascorbate, uric acid, dopamine, catechol, cystine and epinephrine will not interfere with the sensitive determination of H2O2.
Sensors and Actuators B: Chemical.
-
[show abstract]
[hide abstract]
ABSTRACT: In this paper, we present a simple electrochemical method to detect DNA oxidative damage caused by the Fenton’s reagent. With the aid of DNA modified electrode, a electrochemical probe, namely, is used to monitor oxidized damage to DNA. Moreover, it is found that epigallocatechin gallate (EGCG), chlorogenic acid (CGA) and ascorbic acid (AA) have repairing effect to the oxidized DNA, which has also been validated by the proposed technique in this work. The results in this study may not only have implications in determining the chemotherapeutic effects of Chinese traditional medicinal herb species but also have potential use in evaluating their anti-oxidation capacities in oxidative stress related diseases.
Electrochemistry Communications.
