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1.
A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L−1 HCl–0.10 mol L−1 NaCl solution containing 0.10 mmol L−1 Br2. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL−1 to 40 ng mL−1 with a detection limit of 5.2 × 10−10 g mL−1. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.  相似文献   

2.
Jia H  Han X  Li Z  Tian Q  Miao X  Du L  Liu Y 《Talanta》2011,85(4):1871-1875
Accumulating evidence suggests that S-nitrosothiols (RSNOs) play key roles in human health and disease. To clarify their physiological functions and roles in diseases, it is necessary to promote some new techniques for quantifying RSNOs in blood and other biological fluids. Here, a new method using gold nanoparticle catalysts has been introduced for quantitative evaluation of RSNOs in blood serum. The assay involves degrading RSNOs using gold nanoparticles and detecting nitric oxide (NO) released with NO-selective electrodes. The approach displays very high sensitivity for RSNOs with a low detection limit in the picomolar concentration range (5.08 × 10−11 mol L−1, S/N = 3) and is free from interference of some endogenous substances such as NO2 and NO3 co-existing in blood serum. A linear function of concentration in the range of (5.0-1000.0) × 10−9 mol L−1 has been observed with a correlation coefficient of 0.9976. The level of RSNOs in blood serum was successfully determined using the described method above. In addition, a dose-dependent effect of gold nanoparticles on the sensitivity for RSNOs detection is revealed, and thereby the approach is potentially useful to evaluate RSNOs levels in various biological fluids via varying gold nanoparticles concentration.  相似文献   

3.
Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL−1, which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL−1 of hLH and 14 mIU mL−1 of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities <6%, chip-to-chip variabilities <5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.  相似文献   

4.
Yang Y  Wang Z  Yang M  Li J  Zheng F  Shen G  Yu R 《Analytica chimica acta》2007,584(2):268-274
A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO2 and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO2/chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10−10 to 9.32 × 10−8 mol L−1 with the detection limit of 7.5 × 10−11 mol L−1 (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log CDNA (mol L−1) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO2 to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.  相似文献   

5.
Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I50 and detection limits were 0.05-2.5 and 0.1-7.5 μg L−1, 0.35 (±0.04) μg L−1 and 0.9 (±0.1) μg L−1, 60 and 100 μg L−1 in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I50 in real samples was 0.2 μg L−1 corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.  相似文献   

6.
Xiang Y  Mei L  Li N  Tong A 《Analytica chimica acta》2007,581(1):132-136
A new fluorogenic method for the selective and sensitive determination of chromium(VI) in acidic water using rhodamine B hydrazide was developed. This method was based on the oxidation of non-fluorescent rhodamine B hydrazide by potassium dichromate in acidic aqueous conditions to give rhodamine B, which was highly fluorescent, as a product. With the optimum condition described, the fluorescence enhancement at 585 nm was linearly related to the concentration of chromium(VI) in the range of 5.0 × 10−8 to 2.0 × 10−6 mol L−1 (2.60-104 ng mL−1) with a correlation coefficient of R2 = 0.9993 (n = 18) and a detection limit of 5.5 × 10−9 mol L−1 (0.29 ng mL−1). The R.S.D. was 2.2% (n = 5). The proposed method was also applied to the determination of chromium(VI) in drinking water, river water and synthetic samples.  相似文献   

7.
A fast and highly sensitive assay for pepsinogen I (PG I) and pepsinogen II (PG II) by using time-resolved fluoroimmunoassay (TRFIA) detection technique has been developed for the determination of serum PG I and PG II against gastrointestinal diseases. On the noncompetitive assay, one monoclonal antibody (McAb) coated on wells was directed against a specific antigenic site on the PG I or PG II. The McAb, called as labelling McAb, was prepared with the europium-chelate of N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N,N,N-tetraacetic acid and directed against a different antigenic site on the PG I or PG II molecule. After bound/free separation by washing, the fluorescence counts of bound Eu3+-McAb were measured. The levels of PG in sera from patients or healthy volunteers were determined by PG I and PG II TRFIA using the autoDELFIA1235 system. The measurement ranges of PG I-TRFIA were 3.5-328.0 μg L−1 and those of PG II-TRFIA were 2.0-55.0 μg L−1. The within-run and between-run CVs of the PG I-TRFIA were 1.9% and 4.7%, respectively, and those of PG II-TRFIA were 2.1% and 3.8%, respectively. The recovery rates of PG I-TRFIA and PG II-TRFIA were 102.7% and 104.6%, respectively. The detection limitations of PG I and PG II were 0.05 μg L−1 and 0.02 μg L−1, respectively. The dilution experiments showed the percentage of expected value of PG I-TRFIA was 93.2-102.3% and of PG II-TRFIA was 97.3-110.6%. The cross-reacting rate between PG I and PG II was negligible. The linear correlation of radioimmunoassay (RIA) and TRFIA measurements resulted in a correlation coefficient as 0.926 of PG I and as 0.959 of PG II. The europium-labelling McAbs were stable for at least one year at −20 °C, and the results of the TRFIA with same reagents were reproducible over one year as well. The means of 1600 healthy volunteers were 162.4 ± 52.1 μg L−1 for serum PG I, 11.7 ± 6.8 μg L−1 for serum PG II, and 13.8 ± 7.4 for the PG I/PG II ratio. The normal ranges of Serum PG I levels for healthy volunteers were 58.2-266.6 μg L−1, and those of serum PG II levels were less than 25.3 μg L−1. The availability of a highly sensitive, reliable, and convenient PG-TRFIA method for quantifying PG will allow investigations into the possible diagnostic value of this analysis in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastric ulcer and gastritis. The sensitivity and reproducibility of the assay were satisfactory for clinical applications.  相似文献   

8.
Ultrasensitive cysteine sensing using citrate-capped CdS quantum dots   总被引:1,自引:0,他引:1  
Wang GL  Dong YM  Yang HX  Li ZJ 《Talanta》2011,83(3):943-947
The importance of cysteine (Cys) in biological systems has stimulated a great deal of efforts in the development of analytical methods for the determination of this amino acid. In this work, a novel fluorescent probe for Cys based on citrate (Cit)-capped CdS quantum dots (QDs) is reported. The Cit-capped CdS QDs fluorescent probe offers good sensitivity and selectivity for detecting Cys. A good linear relationship was obtained from 1.0 × 10−8 mol L−1 to 5.0 × 10−5 mol L−1 for Cys. The detection limit was calculated as 5.4 × 10−9 mol L−1. The proposed method was applied to detect Cys in human urine samples, which showed satisfactory results. This assay is based on both the lability of Cit and the strong affinity of thiols to the surface of CdS QDs. The addition of Cys improved the passivation of the surface traps of CdS QDs and enhanced the fluorescence intensity.  相似文献   

9.
The development of a direct competitive enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies specific for semicarbazide (SEM) is described. Molecular modelling of the hapten mimics and other key components of the assay system was conducted to explain antibody properties in relation to hapten design. The small aliphatic molecule SEM was coupled to 3-carboxybenzaldehyde to produce carboxyphenyl-SEM (CPSEM), for the generation of specific antibodies. Five rabbits produced antibodies against NPSEM (used in direct and indirect ELISA formats) exhibiting a 50% binding inhibition level (IC50 values) of 0.06-2.28 μg L−1 in assay buffer for SEM. The most sensitive indirect assay based on the antibody MVK39 showed a high dynamic range providing a linear readout in the range of 0.01-0.2 μg L−1. Antibody MVK31 (IgG) allowed specific SEM detection at an IC50 = 0.14 μg L−1 in direct ELISA and was evaluated using solvent extracted SEM-spiked porcine and baby food samples. Recovery levels determined from fortified samples (0.5, 1.0, 1.5, 5, 10 and 20 μg kg−1) of porcine and baby food ranged from 82.9 to 105.3%, respectively, with a coefficient of variation less than 15.5%. Respective detection capability and threshold of the assay for porcine muscle, set on the basis of acceptance of no false negative results, was 0.3 and 0.11 μg kg−1.  相似文献   

10.
An analytical method using an optical probe in a photoelectrochemical cell for the sensitive and selective determination of aqueous Hg2+ is presented. A previously synthesized Hg2+ selective chemosensor, proven to be Hg2+ sensitive up to 2 μg L−1, has been immobilized onto indium tin oxide (ITO) electrodes in a composite form with polyaniline. The coated ITO electrode was placed in a photoelectrochemical cell under closed circuit conditions in which the optical recognition of the chemosensor was converted to a measurable signal. A composite of the fluorescent chemosensor, Rhodamine 6G derivative (RS), and polyaniline (PANI) was immobilized on ITO glass plates and subjected to photovoltage measurements in the absence and presence of Hg2+. The optical responses of the coated electrode were used to determine the sensitivity and selectivity of the immobilized sensor to Hg2+ in the presence of background ions. The optical response of the PANI-dye coated electrode increased linearly with increasing Hg2+ concentration in the range 10-150 μg L−1, with a detection limit of 6 μg L−1.  相似文献   

11.
A new palladium nanoparticle functionalized multi-wall carbon nanotubes (nano-Pd/CNTs) modified pyrolytic graphite electrode (PGE) has been fabricated for electrochemical sensing of calcium dobesilate (CD) in pharmaceutical capsules. The nano-Pd/CNTs were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The nano-Pd/CNTs composite showed a strong electrocatalytic property for CD. The anodic peak current is 6-fold than that obtained in bare PGE and the oxidation potential has an obvious shift to negative. The anodic peak current is proportional to the concentration of CD in the range of 1.0 × 10−7 to 7.0 × 10−4 mol L−1, with a linear relative coefficient r = 0.999 and a detection limit 4.0 × 10−8 mol L−1 (S/N = 3). This kind of electrode shows good stability, sensitivity, reproducibility, large linear range and low detection limit towards electrochemical determination of CD. The proposed method provides a selective and sensitive electrochemical sensor of calcium dobesilate.  相似文献   

12.
A novel method for the determination of peroxynitrite using folic acid as a fluorescent probe is described. The method is based on the oxidation of the reduced, low-fluorescent folic acid by peroxynitrite to produce a high-fluorescent emission product. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 3 × 10−8 to 5.0 × 10−6 mol L−1 with a correlation coefficient of 0.998, and the detection limit is 1 × 10−8 mol L−1. Interferences from some metal ions normally seen in biological samples, and also some anions structurally similar to peroxynitrite were studied. The optimal conditions for the detection of peroxynitrite were evaluated.  相似文献   

13.
Haghighi B  Bozorgzadeh S 《Talanta》2011,85(4):2189-2193
ZnO nanoparticles (nanoZnO) were decorated on multiwalled carbon nanotubes (MWCNTs) and then the prepared nano-hybrids, nanoZnO-MWCNTs, were immobilized on the surface of a glassy carbon electrode (GCE) to fabricate nanoZnO-MWCNTs modified GCE. The prepared electrode, GCE/nanoZnO-MWCNTs, showed excellent electrocatalytic activity towards luminol electrochemiluminescence (ECL) reaction. The electrode was then further modified with lactate oxidase and Nafion to fabricate a highly sensitive ECL lactate biosensor. Two linear dynamic ranges of 0.01-10 μmol L−1 and 10-200 μmol L−1 were obtained for lactate with the correlation coefficient better than 0.9996. The detection limit (S/N = 3) was 4 nmol L−1 lactate. The relative standard deviation for repetitive measurements (n = 6) of 10 μmol L−1 lactate was 1.5%. The fabrication reproducibility for five biosensors prepared and used in different days was 7.4%. The proposed ECL lactate biosensor was used for determination of lactate in human blood plasma samples with satisfactory results.  相似文献   

14.
Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L−1 and 1.3 ng L−1 using two different up-converting phosphors and 0.16 ng L−1 using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.  相似文献   

15.
Suspension array technology for simultaneous detection of three kinds of veterinary drugs, chloramphenicol (CAP), clenbuterol and 17-beta-estradiol has been developed. Conjugates of chloramphenicol and clenbuterol coupled with bovine serum albumin were synthesized and purified. Probes of suspension array were constituted by coupling the three conjugates on the fluorescent microspheres/beads and the microstructures of the beads’ surface were observed by scanning electron microscopy which was a direct confirmation for the successful conjugates’ coupling. The optimal addition of conjugates and the amounts of antibodies were optimized and selected, respectively. Standard curves were plotted and the coefficient of determination-R2 was greater than 0.989 which suggested good logistic correlation. The detection ranges for the three veterinary drugs are 40-6.25 × 105 ng L−1, 50-7.81 × 105 ng L−1 and 1 × 103-7.29 × 105 ng L−1, respectively and the lowest detection limits (LDLs) of them are 40, 50 and 1000 ng L−1, respectively. The suspension array is specific and has no significant cross-reactivity with other chemicals. Meanwhile, unknown samples were detected by suspension array and ELISA in comparison with each other. The errors between found and real for the detection of the unknown samples were relatively small to both of the two methods, whereas, the detection ranges of suspension array are broader and sensitive than that of the traditional ELISA. The high-throughput suspension array is proved to be a novel method for multi-analysis of veterinary drugs with simple operation, high sensitivity and low cost.  相似文献   

16.
A new molecularly imprinted electrochemiluminescence (ECL) sensor was proposed for highly sensitive and selective determination of ultratrace Be2+ determination. The complex of Be2+ with 4-(2-pyridylazo)-resorcinol (PAR) was chosen as the template molecule for the molecularly imprinted polymer (MIP). In this assay, the complex molecule could be eluted from the MIP, and the cavities formed could then selectively recognize the complex molecules. The cavities formed could also work as the tunnel for the transfer of probe molecules to produce sound responsive signal. The determination was based on the intensity of the signal, which was proportional to the concentrations of the complex molecule in the sample solution, and the Be2+ concentration could then be determined indirectly. The results showed that in the range of 7 × 10−11 mol L−1 to 8.0 × 10−9 mol L−1, the ECL intensity had a linear relationship with the Be2+ concentrations, with the limit of detection of 2.35 × 10−11 mol L−1. This method was successfully used to detect Be2+ in real water samples.  相似文献   

17.
An electrochemical DNA detection method for the phosphinothricin acetyltransferase (PAT) gene sequence from the transgenetic plants was established by using a microplate hybridization assay with cadmium sulfide (CdS) nanoparticles as oligonucleotides label. The experiment included the following procedures. Firstly target PAT ssDNA sequences were immobilized on the polystyrene microplate by physical adsorption. Then CdS nanoparticle labeled oligonucleotide probes were added into the microplate and the hybridization reaction with target ssDNA sequences took place in the microplate. After washing the microplate for three times, certain amounts of HNO3 were added into the microplate to dissolve the CdS nanoparticles anchored on the hybrids and a solution containing Cd2+ ion was obtained. At last differential pulse anodic stripping voltammetry (DPASV) was used for the sensitive detection of released Cd2+ ion. Based on this principle a sensitive electrochemical method for the PAT gene sequences detection was established. The voltammetric currents of Cd2+ were in linear range with the target ssDNA concentration from 5.0 × 10− 13 to 1.0 × 10− 10 mol/L and the detection limit was estimated to be 8.9 × 10− 14 mol/L (3σ). The proposed method showed a good promise for the sensitive detection of specific gene sequences with good selectivity for the discrimination of the mismatched sequences.  相似文献   

18.
In this study, a new, sensitive, and rapid assay was developed to quantitatively measure the proteolytic enzyme activity using the surface-enhanced Raman scattering (SERS) probe. Two different shapes of gold nanoparticles, gold nanosphere and nanorod particles were produced. SERS label, comprising self-assembled monolayers (SAMs) of Raman reporter molecule (5,5-Dithiobis (2-Nitrobenzoic acid), DTNB), was coated on the surface of the nanoparticles. Two different SERS-based analysis platforms were designed using gold-coated glass slide and polystyrene microtiter plate. The calibration curves were obtained by plotting the intensity of the SERS signal of symmetric NO2 stretching of DTNB at 1326 cm−1vs. the protease concentration. The effects of nanoparticle geometry and assay platform on the protease assay were investigated and the best working combination of the parameters was selected as rod shaped SERS probe and gold-coated glass slide. The correlation between the protease activity and SERS signal was found to be linear within the range of 0.1-2 mU/mL (R2 = 0.979). The limit of detection (LOD) and limit of quantification (LOQ) values of the validated method were found as 0.43 and 1.30 mU/mL, respectively. The intra-day and inter-day precisions of the method, as relative standard deviation (RSD), were determined as 2.5% and 3.6%, respectively. The developed method was successfully applied for quantitative analysis of the commercial enzyme preparate that is used in cheese making process. It was also used for investigation of substrate specificity of protease enzyme towards the casein and bovine serum albumin. The proposed method has a flexibility to try different substrates for the detection of various enzyme activities.  相似文献   

19.
A simple, specific and sensitive sequential injection analysis (SIA) system based on non-immunoassay fluorescent detection has been developed for the determination of urinary albumin. The specific binding of the dye Albumin Blue 580 (AB 580) to albumin in urine generated high emission fluorescent signals. The excitation and emission wavelengths were set at 590 and 610 nm, respectively. The analytical range was obtained from 1 to 100 mg L−1, with a detection limit of 0.3 mg L−1 (S/N = 3). The SIA system gave high precision with relative standard deviations (R.S.D.s) of 0.9% and 1.4% when evaluated with 15 and 100 mg L−1 albumin (n = 15), respectively. The method exhibited good reproducibility, as assessed by performing four analytical curves on different days, and intra-run CVs (2.3-3.3%) and inter-run CVs (3.8%) were obtained. Rapid operation was achieved with a sample throughput of 37 h−1. This method was successfully applied to the determination of urinary albumin, and the method was highly correlated with the immunoturbidimetric method (r2 = 0.965; n = 72).  相似文献   

20.
We report a low cost selective analytical method based on inner filter effect (IFE) for citrate-silver nanoparticle (cit-AgNP) detection, in which fluorescent amine-derivatized carbon dots (a-CDs) act as the donor and aggregated cit-AgNPs as the energy receptor. Carbon dots (CDs) were chemically modified with ethylenediamine (EDA) moieties via amidic linkage displaying an emission band at 440 nm. The presence of cit-AgNPs produces a remarkably quenching of a-CD fluorescence via IFE, since the free amine groups at CD surface induce the aggregation of cit-AgNPs accompany by a red-shifting of their characteristic plasmon absorption wavelength, which resulted in “turn-on” of the IFE-decreased in CD fluorescence. The proposed method, which involves the use of chelating agents for removal of metal ions interferences, exhibits a good linear correlation for detection of cit-AgNPs from 1.23 × 10−5 to 6.19 × 10−5 mol L−1, with limits of detection (LOD) and quantification (LOQ) of 5.17 × 10−6 and 1.72 × 10−5 mol L−1, respectively. This method demonstrates to be efficient and selective for the determination of cit-AgNPs in complex matrices such as cosmetic creams and reveals many advantages such as low cost, reusability, high sensitivity and non time-consuming compared with other traditional methods.  相似文献   

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