罗丹明B酶联免疫分析方法的建立与应用

建立一种用于检测罗丹明B的间接竞争酶联免疫分析方法。该方法的IC50为1.3ng/m L,检测限为0.786ng/g。该方法检测豆瓣酱,加标浓度5 ng/g,平均回收率为83%,加标浓度10 ng/g,平均回收率为89%;检测辣椒酱,加标浓度5 ng/g,均回收率为88%,加标浓度10 ng/g,平均回收率为104%;检测辣椒油,加标浓度5 ng/g,平均回收率为94%,加标浓度10 ng/g,平均回收率为101%;检测辣椒粉,添加浓度5 ng/g,平均回收率为82%,加标浓度10 ng/g,平均回收率为90%。该方法的平均批内差为9.0%,批间差为10.5%,与其他常见染料未见交叉反应。该方法操作简便,结果准确,适用于罗丹明B的现场大量样品快速检测。     

High sensitivity luminescence nanoparticle assay for the detection of protein aggregation

Nanoparticle assay utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for the detection of protein aggregation. This mix-and-measure nanoparticle assay is based on the competitive adsorption of the sample and the acceptor-labeled protein to donor europium(III) polystyrene particles. The protein aggregation was detected with the developed TR-LRET nanoparticle assay, UV240 absorbance and dynamic light scattering (DLS). All methods well equally detected the aggregation and aggregates, whose size ranged from single protein to more than 1000 nm aggregates. The developed method allowed the aggregation detection of the entire size range at more than 10,000 times lower concentration, 30 μg/L, compared to UV240 and DLS. The simple-to-use and sensitive nanoparticle assay with existing microtiter plate luminometric instrumentation can find use as a routine tool for protein aggregation studies in biochemical laboratories and for quality assessment of protein products in industry.     

黄衣红曲中黄曲霉毒素B_1的酶联免疫检测法

本文报道了ELISA方法测定福建大田县传统工艺生产的酿酒用黄衣红曲中黄曲霉毒素B_1含量。随机抽取了四个生产厂家的样品进行分析,检测结果表明四个黄衣红曲的样品所含黄曲霉毒素B_1含量均低于国家食品安全限量标准,处于安全范围之内。     

Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum hsp70 mRNA

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Cryptosporidium parvum in water has been developed. The target molecule was a 121-nucleotide sequence from the C. parvum heat shock protein 70 (hsp70 mRNA from U71181 gene). This analyte offers the possibility of distinguishing dead from live oocysts. The assay involves covalent attachment of a primary DNA probe via its 5'-amine-terminus to self-assembled monolayers of mercaptoundecanoic acid to a gold surface. The primary DNA probe was used to capture the target (sequence 1039-1082 of U71181 gene for the mRNA), by hybridization to a 20-base complementary sequence on the target (at sequence 1063-1082). A secondary DNA probe labeled with alkaline phosphatase (AP) was then hybridized to base sequence 1039-1062 on the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution, in which gold-coated silicon wafer modified with the complete assembly of the assay components was incubated, is linear with concentration of the target (5-50 microg/mL, where P1 and P2-AP concentrations are 50 microg/mL). A detection limit of 2 microg/mL (or 146 nM) of the DNA target was obtained. Cross-reactivity tests showed high selectivity for heat-shocked C. parvum. No signal was obtained for either the synthetic DNA for hsp70 of Campylobacter lari, Escherichia coli, Giardia lamblia, Salmonella typhimurium, and Listeria monocytogenes or for the products of heat-shocked whole organisms of E. coli, G. lamblia, Staphylococcus aureus, and Cryptosporidium muris.     

Plasmon-enhanced colorimetric ELISA with single molecule sensitivity

Robust but ultrasensitive biosensors with a capability of detecting low abundance biomarkers could revolutionize clinical diagnostics and enable early detection of cancer, neurological diseases, and infections. We utilized a combination of localized surface plasmon resonance (LSPR) refractive index sensing and the well-known enzyme-linked immunosorbent assay to develop a simple colorimetric biosensing methodology with single molecule sensitivity. The technique is based on spectral imaging of a large number of isolated gold nanoparticles. Each particle binds a variable number of horseradish peroxidase (HRP) enzyme molecules that catalyze a localized precipitation reaction at the particle surface. The enzymatic reaction dramatically amplifies the shift of the LSPR scattering maximum, λ(max), and makes it possible to detect the presence of only one or a few HRP molecules per particle.     

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant

Three haptens of the organophosphorus (OP) toxicant ‘sarin’ having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC₅₀ (inhibitory concentration) of free antigen was found to be 0.415 μg mL⁻¹ on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19-1.56 μg mL⁻¹ with a limit of detection (LOD) of 0.05 μg mL⁻¹. The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.     

High sensitivity DNA detection based on regioselectively decorated electrocatalytic nanoparticles

Self-assembled monolayers (SAMs) of dodecanethiol have been formed on gold electrodes to produce nanoscale defects. These defects define nucleation sites for the electrodeposition of mushroom shaped platinum nanoparticles (PtNPs). The top surfaces of these PtNPs have been selectively functionalized with single stranded probe DNA. These regioselectively modified particles were desorbed by applying a current jump to yield nanoparticles capable of biorecognition on the top curved side and efficient electrocatalysis on the nonfunctionalized lower surface. A second electrode was functionalized with single stranded capture DNA that has a sequence that is complementary to the pathogen, Staphylococcus aureus but leaves a section of the target available to bind the probe strand immobilized on the PtNPs. Following hybridization of the target and capture strands, the surface was exposed to the probe DNA labeled electrocatalytic PtNPs. Target binding was detected by monitoring the current associated with the reduction of hydrogen peroxide in a solution of 0.01 M H(2)SO(4). Calibration plots of the log[DNA] versus faradaic current were linear from 10 pM to 1 μM and picomolar concentrations could be detected without the need for amplification of the target, for example, using PCR or NASBA. As well as a wide dynamic range, this detection strategy has an excellent ability to discriminate DNA mismatches and a high analytical sensitivity.     

Simple bead assay for detection of live bacteria (Escherichia coli)

Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.     

Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers

A novel flow-through immunosensing system for performing a multianalyte chemiluminescent determination in a single run was designed. A new analytical strategy of substrate zone-resolved technique was proposed. Using carcinoma antigen 125 (CA 125) and carcinoembryonic antigen (CEA) as model analytes, the capture antibodies for CA 125 and CEA were immobilized on an UltraBind aldehyde-activated membrane to act as an immunoreactor, to which the mixture of CA 125, CEA, and their corresponding tracers, horseradish peroxidase (HRP)-labeled anti-CA 125 and alkaline phosphatase (ALP)-labeled anti-CEA, was introduced for on-line incubation. The substrates for HRP and ALP were then delivered into the detection cell sequentially to perform substrate zone-resolved immunoassay by a sandwich format. Under optimal conditions, CA 125 and CEA could be assayed in the ranges of 5.0-100 units/mL and 1.0-120 ng/mL, respectively. The whole assay process including incubation, wash, detection, and regeneration could be completed in 35 min. The serum samples from the clinic were assayed with the proposed method, and the results were in acceptable agreement with the reference values. This method and the strategy of substrate zone-resolved technique could be further developed for high-throughput multianalyte immunoassay.     

Heterogeneous immunosensing using antigen and antibody monolayers on gold surfaces with electrochemical and scanning probe detection

We report the use of antibody and antigen monolayer immunosurfaces as detection elements in a competitive heterogeneous immunoassay employing either electrochemical or scanning probe detection. Antibody or antigen monolayers were prepared by covalent attachment of the desired immunoreagent to a two-component self-assembled monolayer via amide linkages. More specifically, mixed monolayers of a carboxylic acid-terminated thiol (thioctic acid) and a methyl-terminated thiol (butanethiol) were used to control the surface epitope density. The microscopic structure of the resulting antibody and antigen arrays was characterized by AFM (atomic force microscopy). Individual, surface-confined rabbit IgG antibodies could be directly imaged in contact mode. The average height of the capture antibodies was found to be 7.1 nm; the average antibody diameter, after correcting for tip convolution effects, was determined to be between 7 and 10 nm. The surface epitope density could be varied over approximately 2 orders of magnitude by changing the composition of the mixed monolayer. AFM was also used to characterize the antibody-antigen binding characteristics of these immunosurfaces, and an average binding efficiency of 22.8% was measured for rabbit IgG antibody arrays. In the second part of this study, the electrochemical detection scheme originally developed by Heineman and co-workers was adapted to our system. A calibration data set was measured, and the linear least-squares correlation coefficient (R2) was found to be 0.993. Finally, the electrochemical and scanning probe detection modes were directly compared. We find an excellent correlation between the surface density of antibody-antigen complexes measured by AFM and the electrochemical response of the same immunosurfaces.     

Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins

A simple and rapid homogeneous enzyme-linked binding assay method for studying lectin-carbohydrate interactions is described. The method is based on the homogeneous inhibition of appropriate enzyme-saccharide conjugates by specific carbohydrate-binding lectins. In the presence of carbohydrate structures recognized by the lectins, enzyme activity is regained in an amount of proportional to the concentration of carbohydrate. The new method can be used to rapidly assess the relative carbohydrate specificity of the various lectins and for the selective analytical detection of simple saccharides and complex glycoproteins. Indeed, when Jacalin lectin is used in conjunction with a malate dehydrogenase-galactose conjugate, selective measurement of human IgA (immunoglobulin A) at microgram per milliliter levels in less than 10 min is possible. The potential for using this analytical methodology for determining changes in the carbohydrate structure of intact recombinant glycoproteins is also discussed.     

Achieving high detection sensitivity (14 zmol) of biomolecular ions in bioaerosol mass spectrometry

Bioaerosol mass spectrometry (BAMS) performs single-cell analysis in real time. However, the specificity of BAMS mass signatures has been limited by low sensitivity at high masses. To increase the mass range and sensitivity of BAMS, a novel design was developed that utilizes a linear flight tube with delayed extraction and an electrostatic ion guide. This study quantifies the sensitivity limits of the novel BAMS design and evaluates the feasibility of BAMS to detect higher mass biomarkers from single cells. All experiments were carried out using MALDI aerosol particles that were nebulized from solution. Sensitivity was assessed by generating particles with decreasing amounts of analyte via serial dilutions. The amount of analyte contained within each particle was calculated based on particle size, density, and molarity of the analyte within solution. A variety of biomolecular ions were studied and signals obtained from particles containing 300 zmol of maltopentaose, 132 zmol of alpha-cyclodextrin, and 14 zmol (approximately 8400 molecules) of gramicidin S are reported. The detection of 14 zmol of gramicidin S is to the best of our knowledge a record in sensitivity for MALDI TOF-MS.     

High sensitivity noise measurement with a correlation spectrumanalyzer

A spectrum analyzer with enhanced sensitivity has been built and used in noise measurements. It is based on the processing of the input signal by two independent channels in parallel and takes advantage of the incoherent property of the noise in each of the two input stages. The instrument has demonstrated an improvement in sensitivity of at least 50 dB with respect to a traditional system, and therefore can measure low input signals down to the hundred pV/√Hz range     

Lead (II) ion detection in surface water with pM sensitivity using aza-crown-ether-modified silver nanoparticles via dynamic light scattering

In this paper, we reported ultrasensitive lead ion detection in environmental water with pM sensitivity using aza-crown-ether-modified silver nanoparticles (ACE-Ag NPs) through dynamic light scattering (DLS). The colorimetric method based on ACE-Ag NPs is not capable of detecting Pb2+ ions over other metal ions (Fe3+, Co2+, Cu2+, Hg2+, Cd2+, Ag+, Li+, Na+, K+ and Cs+) with high sensitivity. But DLS has improved the selectivity and sensitivity of the Pb2+ detection system (50-fold or more) over colorimetric method, and its detection limit is 0.25 pM (1.03 ppt). The Pb2+ DLS assay can be applied to detect Pb2+ in the environmental water, such as in Yangtze and East Lake water samples with a detection limit of 0.20 and 0.22 pM, which is much lower than the maximum contamination level of 4.8×10(-8) M for lead in surface water defined by the national environmental quality standards of China (GB 3838-2002). Also, this method has a good performance in the determination of Pb2+ in drinking water, which is much lower than the maximum contamination level (MCL) of 72 nM for lead as defined by the US Environmental Protection Agency (EPA).     

Taqman MGB探针快速定量检测VHSV方法的研究

为建立准确实时地定量检测病毒性出血性败血症病毒(VHSV),在VHSV-N基因保守区设计了Taqman MGB探针与引物对,随后,采用体外转录技术获得了VHSV-N基因RNA,并以此为绝对定量标准品,建立了绝对定量(AQ)检测VHSV的实时荧光RT-PCR法(AQ-RT-PCR方法),并与世界动物卫生组织(OIE)推荐的普通RT-PCR法进行了比较。此荧光RT-PCR法特异性好,与其他鱼类弹状病毒无交叉反应。检测线性范围为10~(10)～10~2拷贝/反应,灵法度达10~2拷贝/反应。此检测灵敏度比OIE推举的RT-PCR法高出5个数量级,比嵌套RT-PCR高出1个数量级。此法是出入境检疫VHSV的有效方法。     

High sensitivity fiber optic angular displacement sensor and its application for detection of ultrasound

In this paper, we report on the development of an intensity-modulated fiber-optic sensor for angular displacement measurement. This sensor was designed to present high sensitivity, linear response, and wide bandwidth and, furthermore, to be simple and low cost. The sensor comprises two optical fibers, a positive lens, a reflective surface, an optical source, and a photodetector. A mathematical model was developed to determine and simulate the static characteristic curve of the sensor and to compare different sensor configurations regarding the core radii of the optical fibers. The simulation results showed that the sensor configurations tested are highly sensitive to small angle variation (in the range of microradians) with nonlinearity less than or equal to 1%. The normalized sensitivity ranges from (0.25×V(max)) to (2.40×V(max)) mV/μrad (where V(max) is the peak voltage of the static characteristic curve), and the linear range is from 194 to 1840 μrad. The unnormalized sensitivity for a reflective surface with reflectivity of 100% was measured as 7.7 mV/μrad. The simulations were compared with experimental results to validate the mathematical model and to define the most suitable configuration for ultrasonic detection. The sensor was tested on the characterization of a piezoelectric transducer and as part of a laser ultrasonics setup. The velocities of the longitudinal, shear, and surface waves were measured on aluminum samples as 6.43, 3.17, and 2.96 mm/μs, respectively, with an error smaller than 1.3%. The sensor, an alternative to piezoelectric or interferometric detectors, proved to be suitable for detection of ultrasonic waves and to perform time-of-flight measurements and nondestructive inspection.     

伊藤型模糊随机微分方程

提出了伊藤(Ito)型模糊随机微分方程的概念,证明了其解的存在唯一性,给出伊藤型线性模糊随机微分方程解的表达式和统计特征方程,并通过例子说明了解法。     

Automated detection of irradiated food with the comet assay

Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose-response curves, and hence a sufficiently accurate dose estimation.