共查询到20条相似文献,搜索用时 46 毫秒
1.
Christopher W. Heppel Anne-Kathrin Heling Elmar Richter 《Analytical and bioanalytical chemistry》2009,393(5):1525-1530
4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific
nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on
Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3–2.0 g of human target tissues
such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific
method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative
chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of
14.9 fmol HBP and a recovery of 45 ± 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of
esophageal mucosa (N = 14) yielding 5.6 ± 1.9 mg tissue and a mean adduct level of 6.13 ± 9.35 pmol HPB/mg DNA. 相似文献
2.
Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine 总被引:1,自引:0,他引:1
Francesco Zezza Francesco Longobardi Michelangelo Pascale Sergei A. Eremin Angelo Visconti 《Analytical and bioanalytical chemistry》2009,395(5):1317-1323
A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer,
has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl
solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and
5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min.
The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and
compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation
(r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for
70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels
close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed
FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for
white and rosé wines, which are known to contain less interfering compounds such as polyphenols. 相似文献
3.
A kind of erbium hexacyanoferrate (ErHCF)-modified carbon ceramic electrodes (CCEs) fabricated by mechanically attaching ErHCF
samples to the surface of CCEs derived from sol–gel technique was proposed. The resulting modified electrodes exhibit well-defined
redox responses with the formal potential of +0.215 V [vs saturated calomel electrode (SCE)] at a scan rate of 20 mV s−1 in 0.5 M KCl (pH 7) solution. The voltammetric characteristics of the ErHCF-modified CCEs were investigated by voltammetry.
Attractively, the ErHCF-modified CCEs presented good electrocatalytic activity with a marked decrease in the overvoltage about
400 mV for l-cysteine oxidation. The calibration plot for l-cysteine determination was linear at 5.0 × 10−6–1.3 × 10−4 M with a linear regression equation of I(A) = 0.558 + 0.148c (μM) (R
2 = 0.9989, n = 20), and the detection limit was 2 × 10−6 M (S/N = 3). At last, the ErHCF-modified CCEs were used for amperometric detection of l-cysteine in real samples. 相似文献
4.
Karina B. Hueso Domínguez Ildikó V. Tóth M. Renata S. Souto Filipa Mendes Cándido García De María Isabel Vasconcelos António O. S. S. Rangel 《Applied biochemistry and biotechnology》2010,160(6):1664-1673
A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its
reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD+ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of
glycerol between 0.046 and 0.46 g/l, (corresponding to yeast fermentation samples with concentrations up to 50 g/l) with good
repeatability (relative standard deviation for n = 10 lower than 2.2% for three different samples) at a sampling frequency of 25/h. The detection and quantification limits
using a miniaturised spectrophotometer were 0.13 and 0.44 mM, respectively. Reagent consumption was of 0.45 μmol NAD+ and 1.8 μg enzyme per assay, and the waste production was 2.8 ml per determination. Results obtained for samples were in
agreement with those obtained with a high-performance liquid chromatography method. 相似文献
5.
Herranz S Ramón-Azcón J Benito-Peña E Marazuela MD Marco MP Moreno-Bondi MC 《Analytical and bioanalytical chemistry》2008,391(5):1801-1812
Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N′-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins.
Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine
structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for
simazine (detection limit 0.11 ± 0.02 μg L−1, IC50 0.88 ± 0.04 μg L−1), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated
assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC50 value of 0.35 ± 0.04 μg L−1 with a detection limit of 1.3 ± 0.9 ng L−1 and a dynamic range from 0.010 to 7.5 μg L−1 simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as
atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity
was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as
a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could
be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to
the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative
analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence. 相似文献
6.
Skalka N Krol A Schlesinger H Altstein M 《Analytical and bioanalytical chemistry》2011,400(10):3491-3504
The present research focused on the development of an immunoassay and an immunochemical sol–gel-based immunoaffinity purification
(IAP) method for purification and detection of the non-steroid anti-inflammatory drug (NSAID) indomethacin (IMT). A polyclonal
antibody (Ab) for IMT was generated, and two sensitive microplate assays for the detection of IMT were developed (termed OV
and HRP formats), based on the enzyme-linked immunosorbent assay (ELISA) method. The limits of detection of the assays were
15 ± 1.25 ng mL−1 (n = 50) and 12 ± 0.17 ng mL−1 (n = 4) for the OVA and HRP formats, respectively. The Abs exhibited slight cross-reactivity with other NSAIDs. The Abs were
also used to develop a sol–gel-based IAP method for clean-up and concentration of IMT. Several sol–gel formats with various
amounts of antibodies were examined; the best and most reproducible format was at a TMOS:HCl molar ratio of 1:6 in which 120 μL
of IMT Abs was entrapped. The binding capacity under these conditions was ca. 100 to 250 ng of IMT with very low non-specific
binding (less than 5% of the applied amount). The sol–gel IAP method, combined with solid-phase extraction, successfully eliminated
serum interference to a degree that enabled analysis of spiked serum samples by ELISA. The method was also found to be fully
compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry. The approaches
developed in this study form a basis for analysis of IMT in biological samples in order to monitor their pharmacokinetic properties,
and may be further used to study population exposure to IMT, and to monitor the occurrence of IMT contamination in water samples. 相似文献
7.
Shao-Wen Zhang Jun Xing Ling-Shuang Cai Cai-Ying Wu 《Analytical and bioanalytical chemistry》2009,395(2):479-487
Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of
8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel
molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as
the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and
determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency
owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG
was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise
ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIP’s selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the
proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly
developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer
patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people. 相似文献
8.
Real-time immuno-PCR assay for detecting PCBs in soil samples 总被引:1,自引:0,他引:1
A fast and robust assay, based on immuno-polymerase chain reaction (IPCR) techniques, was developed for the detection of polychlorinated
biphenyls (PCBs) in soil samples. Real-time IPCR (rt-IPCR) is a powerful technique that combines enzyme-linked immunosorbent
assay (ELISA) with the specificity and sensitivity of PCR. In our assay, indirect ELISAs based on immobilization of PCB37
hapten–ovalbumin conjugates was used for evaluation of the immune response. The effect of optimal reagent concentrations to
reduce background fluorescence was also investigated. Using the optimized assay, the linear sensitivity range of the assay
covered more than six orders of magnitude, and the minimum detection limits reached 5 fg ml–1 antigen. Rt-IPCR was tested for its cross-reactivity profiles using four selected congeners and four Aroclor products. The
assays were highly specific for congeners but less specific for Aroclor1242. We took four soil samples to validate the method,
and the results were confirmed by gas chromatography/mass spectrometry (GC/MS). The rt-IPCR results for soil samples correlated
well with the concentrations of PCBs obtained by GC/MS (r = 0.99, n = 6). These data indicate that this highly specific, sensitive, and robust assay can be modified for detecting PCB compounds
in the environment. 相似文献
9.
Giovannoli C Anfossi L Baggiani C Giraudi G 《Analytical and bioanalytical chemistry》2008,392(3):385-393
Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced
fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate
by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation
conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 106 M−1 and (9.06 ± 5.7) × 106 M−1—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow
use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L−1). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising
results in detecting the toxin in complex real matrices. 相似文献
10.
A compact miniaturized continuous flow system for the determination of urea content in milk 总被引:1,自引:0,他引:1
Willian Toito Suarez Osmundo Dantas Pessoa-Neto Vagner Bezerra dos Santos Ana Rita de Araujo Nogueira Ronaldo Censi Faria Orlando Fatibello-Filho Mar Puyol Julián Alonso 《Analytical and bioanalytical chemistry》2010,398(3):1525-1533
A multicommutation-based flow system with photometric detection was developed, employing an analytical microsystem constructed
with low temperature co-fired ceramics (LTCC) technology, a solid-phase reactor containing particles of Canavalia ensiformis DC (urease source) immobilized with glutaraldehyde, and a mini-photometer coupled directly to the microsystem which monolithically
integrates a continuous flow cell. The determination of urea in milk was based on the hydrolysis of urea in the solid-phase
reactor and the ammonium ions produced were monitored using the Berthelot reaction. The analytical curve was linear in the
urea concentration range from 1.0 × 10−4 to 5.0 × 10−3 mol L−1 with a limit of detection of 8.0 × 10−6 mol L−1. The relative standard deviation (RSD) for a 2.0 × 10−3 mol L−1 urea solution was lower than 0.4% (n = 10) and the sample throughput was 13 h−1. To check the reproducibility of the flow system, calibration curves were obtained with freshly prepared solutions on different
days and the RSD obtained was 4.7% (n = 6). Accuracy was assessed by comparing the results of the proposed method with those from the official procedure and the
data are in close agreement, at a 95% confidence level. 相似文献
11.
Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial
infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against
neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins.
The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody
reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 μg L−1, respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material
availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 μg L−1. The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with
sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations
ranging from 3.2 to 103 μg L−1 could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory
correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5–16 μg L−1 (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into
multianalyte biochip detection systems. 相似文献
12.
Wang J Li H Shelver WL Wang Z Li QX Li J Xu T 《Analytical and bioanalytical chemistry》2011,401(7):2249-2258
A sensitive direct enzyme-linked immunosorbent assay (ELISA) for the specific detection of 2,2′,4,4′-tetrabromodiphenyl ether
(BDE-47) in environmental samples was developed. A hapten mimicking BDE-47 was synthesized by introducing a butyric acid spacer
into 5-hydroxy-BDE-47 and coupled to keyhole limpet hemocyanin to form an immunogen for the production of monoclonal antibodies
(Mabs) against BDE-47. The most sensitive direct ELISA was formatted with a Mab, designated as 4F2, in combination with 5-(2,4-dibromophenoxy)pentanoic
acid peroxidase as a tracer. The inhibition half-maximum concentrations and limit of detection of BDE-47 in phosphate buffered
saline with 25% DMSO were 1.4 ± 0.05 and 0.1 ng mL−1, respectively. Cross-reactivity values of the ELISA with the tested BDE congeners and metabolites were ≤5.8%. This assay
was used to determine BDE-47 in soil, sediment and house dust samples after ultrasonic extraction, simple cleanup and concentration
steps. The average recoveries, repeatabilities (intraday extractions and analyses), and intra-laboratory reproducibilities
(interday extractions and analyses) were in a range of 92–126%, 8–19% and 9–25%, respectively. Applied to 44 real samples,
the results of this assay displayed a statistically significant correlation with those of a gas chromatography–mass spectrometry
method (R
2 = 0.79-0.85), indicating this ELISA is a suitable tool for environmental analyses of BDE-47. 相似文献
13.
Shafiquzzaman Siddiquee Nor Azah Yusof Abu Bakar Salleh Soon Guan Tan Fatimah Abu Bakar 《Journal of Solid State Electrochemistry》2012,16(1):273-282
Electrochemical DNA biosensor was successfully developed by depositing the ionic liquid (e.g., 1-ethyl-3-methylimidazolium
trifluoromethanesulfonate ([EMIM][Otf])), ZnO nanoparticles, and chitosan (CHIT) nanocomposite membrane on a modified gold
electrode (AuE). The electrochemical properties of the [EMIM][Otf]/ZnO/CHIT/AuE for detection of DNA hybridization were studied.
Under optimal conditions using cyclic voltammetry, the target DNA sequences could be detected in the concentration range of
1.0 × 10−18 to 1.82 × 10−4 mol L−1, and with the detection limit of 1.0 × 10−19 mol L−1. This DNA biosensor detection approaches provide a quick, sensitive, and convenient method to be used in the identification
of Trichoderma harzianum. 相似文献
14.
Zhouping Wang Nuo Duan Xu Hun Shijia Wu 《Analytical and bioanalytical chemistry》2010,398(5):2125-2132
A highly selective electrochemiluminescent biosensor for the detection of target nephrotoxic toxin, ochratoxin A (OTA), was
developed using a DNA aptamer as the recognition element and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as the signal-producing compound. The electrochemiluminescent aptamer biosensor was fabricated by
immobilizing aptamer complementary DNA 1 sequence onto the surface of a gold-nanoparticle (AuNP)-modified gold electrode.
ABEI-labeled aptamer DNA 2 sequence hybridized to DNA 1 and was utilized as an electrochemiluminescent probe. A decreased
electrochemiluminescence (ECL) signal was generated upon aptamer recognition of the target OTA, which induced the dissociation
of DNA 2 (ABEI-labeled aptamer electrochemiluminescent probe) from DNA 1 and moved it far away from the electrode surface.
Under the optimal conditions, the decreased ECL intensity was proportional to an OTA concentration ranging from 0.02 to 3.0 ng mL-1, with a detection limit of 0.007 ng mL-1. The relative standard deviation was 3.8% at 0.2 ng mL-1 (n = 7). The proposed method has been applied to measure OTA in naturally contaminated wheat samples and validated by an official
method. This work demonstrates the combination of a highly binding aptamer with a highly sensitive ECL technique to design
an electrochemiluminescent biosensor, which is a very promising approach for the determination of small-molecule toxins. 相似文献
15.
Motoi Oishi Hisato Hayashi Keiji Itaka Kazunori Kataoka Yukio Nagasaki 《Colloid and polymer science》2007,285(9):1055-1060
A pH-responsive PEGylated nanogel was successfully prepared by means of emulsion copolymerization of 2-(N,N-diethylamino)ethyl methacrylate (AMA) with heterobifunctional poly(ethylene glycol) (PEG) bearing a 4-vinylbenzyl group at
the α-end and a lactose moiety at the ω-end in the presence of potassium persulfate and ethyleneglycol dimethacrylate as a
cross-linker. Polyplex micelle composed of PEG-block-poly(l-lysine) copolymer and plasmid DNA (PEG-b-PLL/pDNA) exhibited a far more efficient transfection ability in the presence of lac-nanogel-8k-1.0% (PEG, M
n = 8000; cross-linking density, 1.0%) than the PEG-b-PLL/pDNA polyplex micelle alone (in the absence of lac-nanogel-8k-1.0%), suggesting that an appreciable fraction of lac-nanogel-8k-1.0%
along with the PEG-b-PLL/pDNA polyplex micelle is taken up into the HuH-7 cells through the asialoglycoprotein receptor-mediated endocytosis process
mediated by the cluster of a large number of lactose moieties on the surface of lac-nanogel-8k-1.0%, followed by the effective
disruption of the endosome by the buffer effect of the unprotonated PAMA core in lac-nanogel-8k-1.0%.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
A sensitive LC-MS/MS assay for quantification of total and free concentrations of R- and S-warfarin in plasma was required
to support clinical studies on warfarin enantiomers. Several ultrafiltration devices were evaluated for separation of free
warfarin from plasma proteins. The highest precision and lowest non-specific binding was obtained for Centrifree ultrafiltration
devices. R- and S-warfarin were extracted from plasma (total) and ultrafiltrate (free) by liquid–liquid extraction with methyl
tert-butyl ether using d6-warfarin as internal standard. Mean extraction recovery was 68 ± 4%. The enantiomers were separated on a Chirobiotic V column
with isocratic elution using 40% methanol and 0.03% acetic acid in water. Negative mode electrospray ionisation was used for
MS/MS detection, monitoring the ion transition m/z 307/161. Calibration curves (quadratic, weighted 1/x) were fitted over the range of 20–2,000 ng/ml (r
2 ≥ 0.995) in plasma and 0.5–20 ng/ml (r
2 ≥ 0.998) in ultrafiltrate. The lower limit of quantification for R- and S-warfarin was 0.5 ng/ml in ultrafiltrate. Intra-
and interday precision (% RSD) and bias were within 10% in all cases, and matrix effects were negligible. The assay was applied
successfully to analysis of samples from clinical studies. 相似文献
17.
It was found that tripropylamine (TPA) could be used as a coreactant to initiate the electrochemiluminescence (ECL) of acridinium
NHS ester (AE NHS) labels attached to DNA. The radicals generated in the electro-oxidation process of TPA reacted with AE
NHS to form the excited N-methylacridone, giving rise to light emission. The AE/TPA ECL system was for the first time used as the detection system
for developing an ECL-based DNA sensor. In the protocol, streptavidin-modified gold nanoparticles were firstly immobilized
onto a thiol-treated gold electrode. The streptavidin could specifically interact with the biontinylated capture DNA. Afterwards,
the target DNA and the AE-labeled report DNA were conjugated onto the electrode step by step due to the hybridization reactions,
and a sandwich-type sensor was fabricated. The ECL signals of the sensor were obtained under pulse potential condition in
alkaline solution containing 50.0 mmol L−1 TPA. Under optimized experimental conditions, the linear range of the DNA sensor for the determination of the target DNA
was from 5.0 × 10−15 to 5.0 × 10−12 mol L−1. The detection limit (S/N = 3) was 3.0 × 10−15 mol L−1. Moreover, the sensor could specifically recognize the target DNA against one base-pair mismatched sequences, two base-pair
mismatched sequences, and the noncomplementary sequences. It is of great application potential in clinic analysis. 相似文献
18.
S. Schumacher R. Wüstneck B.-R. Paulke R. Cartier U. Pison 《Colloid and polymer science》2009,287(2):203-214
The binding of Gd3+ to two carboxylated polyglycidyl methacrylate latices was investigated. The latices differed in size (60 and 140 nm for CL6
and CL3, respectively) and surface charge density. The Gd3+ concentration in aqueous suspension was determined using an arsenazo (III) assay. Using 153Gd3+, the bound amount was determined directly. Because of the high binding affinity, ligand depletion became evident. The binding
was pH dependent, investigated in buffer solutions not influencing the arsenazo (III) assay. Optimal binding occurs by formation
of sodium salts of the carboxylic groups and replacement of Na+ and H+ by Gd3+. The dissociation constants of the particles were k
D ≈ 5 × 10−5 mol/L (CL3) and 10−4 mol/L (CL6), without cooperativity (Hill plot). Colloidal stability was investigated.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
19.
Jong Il Lee Yun Jae Kim Heejin Bae Sung Suk Cho Jung-Hyun Lee Suk-Tae Kwon 《Applied biochemistry and biotechnology》2010,160(6):1585-1599
The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase
chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column
chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 °C, respectively. The
optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl2, 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 × 10−6) than Taq DNA polymerase (12.13 × 10−6) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time. 相似文献
20.
Bagheri H Aghakhani A Akbari M Ayazi Z 《Analytical and bioanalytical chemistry》2011,400(10):3607-3613
A micro-solid phase extraction technique was developed using a novel polypyrrole-polyamide nanofiber sheet, fabricated by
electrospinning method. The applicability of the new nanofiber sheet was examined as an extracting medium to isolate malathion
as a model pesticide from aqueous samples. Solvent desorption was subsequently performed in a microvial, and an aliquot of
extractant was injected into gas chromatography–mass spectrometry. Various parameters affecting the electrospinning process
including monomer concentration, polyamide content, applied voltage, and electrospinning time were examined. After fabricating
the most suitable preparation conditions, influential parameters on the extraction and desorption processes were optimized.
The developed method proved to be rather convenient and offers sufficient sensitivity and good reproducibility. The limit
of detection (S/N = 3) and limit of quantification (S/N = 10) of the method under optimized conditions were 50 and 100 ng L−1, respectively. The relative standard deviation at concentration level of 1 ng mL−1 was 2% (n = 3). The calibration curve of analyte showed linearity in the range of 0.1–1 ng mL−1 (R
2 = 0.9975). The developed method was successfully applied to tap and Zayanderood river water samples, while the relative recovery
percentages of 98% and 96% were obtained, respectively. The whole procedure showed to be conveniently applicable and quite
easy to be manipulated. 相似文献