Rapid detection of Escherichia coli contamination in packaged fresh spinach using hyperspectral imaging

A rapid method based on hyperspectral imaging for detection of Escherichia coli contamination in fresh vegetable was developed. E. coli K12 was inoculated into spinach with different initial concentrations. Samples were analyzed using a colony count and a hyperspectroscopic technique. A hyperspectral camera of 400-1000 nm, with a spectral resolution of 5 nm was employed to acquire hyperspectral images of packaged spinach. Reflectance spectra were obtained from various positions on the sample surface and pretreated using Sawitzky-Golay. Chemometrics including principal component analysis (PCA) and artificial neural network (ANN) were then used to analyze the pre-processed data. The PCA was implemented to remove redundant information of the hyperspectral data. The ANN was trained using Bayesian regularization and was capable of correlating hyperspectral data with number of E. coli. Once trained, the ANN was also used to construct a prediction map of all pixel spectra of an image to display the number of E. coli in the sample. The prediction map allowed a rapid and easy interpretation of the hyperspectral data. The results suggested that incorporation of hyperspectral imaging with chemometrics provided a rapid and innovative approach for the detection of E. coli contamination in packaged fresh spinach.     

基于间接竞争原理的流式微球技术快速检测麦芽中赭曲霉毒素A

建立了一种快速、灵敏检测中药麦芽中赭曲霉毒素A(Ochratoxin A,OTA)含量的间接竞争流式微球方法.麦芽样品经60％甲醇/PBS提取,加入20％甲醇/PBS溶液稀释5倍后,离心取上清液制备样品.在编码荧光微球上偶联牛血清白蛋白-OTA(BSA-OTA)复合物,与样品中OTA竞争结合抗OTA特异性抗体,然后加入FITC-IgG孵育,离心洗涤后,用流式细胞仪检测微球表面的平均荧光强度,实现样品中OTA的准确定性定量测定.本方法检测OTA的半数抑制浓度IC50为1.20 ng/mL,相关系数R2=0.9892,检出限(IC10)为0.12 ng/mL,加样回收率为93.9％ ～ 97.4％,RSD＜3.6％.16份实际麦芽样品中检测到2个阳性样品,OTA的最高含量为3.83 μg/kg,未超出欧盟的OTA限量标准,与液相色谱-串联质谱(LC-MS/MS)法检测结果相一致.本方法简单、快速、灵敏、可靠,可拓展用于其它复杂基质中多种真菌毒素的定性与定量检测.     

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90–120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

铂纳米颗粒修饰电极对大肠杆菌的电化学快速检测

本文采用了电化学沉积法制备了铂纳米颗粒化学修饰电极（PtNP/GCE）,并将它应用于大肠杆菌的检测。原理是基于检测大肠杆菌溶液中酶与底物的反应产物,对氨基酚,实现了对大肠杆菌的快速检测。采用了铂纳米颗粒修饰电极,并对检测系统进行优化,提高大肠杆菌的检测灵敏度。大肠杆菌浓度在50—1.0×10⁵cfu/ml与响应电流成良好的线性关系,最低检测限为20 cfu/ml,检测时间在4个小时以内。与传统方法相比,该电化学方法能很好地满足食品安全、环境监控和临床医学等领域中快速检测的要求。     

Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen

An electrochemical biosensor for the specific detection of short DNA sequences from the E. coli pathogen is described. This hybridization device relies on the immobilization of a 25-mer oligonucleotide probe, from the E. coli lacZ gene, onto a screen-printed carbon electrode. Chronopotentiometric detection of the Co(bpy)₃⁺³ indicator is used for monitoring the hybridization event. Numerous variables of the assay protocol, including those of the probe immobilization step, the hybridization event, and the indicator association/detection, are characterized and optimized. Hybridization times of 2- and 30-min are sufficient for detecting 300- and 50 ng/mL, respectively, of the E. coli DNA target. Applicability to analysis of untreated environmental water samples is illustrated. Such single-use electrochemical sensors hold great promise for decentralized environmental and food testing for the E. coli pathogen.     

基于微流控技术的磁免疫荧光法在EB病毒检测中的应用

EB病毒(Epstein-Barr virus,EBV)的早期诊断能够降低患者发生重大疾病的风险.临床上常用的EBV抗体的检测方法存在耗时长、试剂消耗大和效率低等缺点.相比于传统的检测方法,微流控(microfluidics)技术具有高通量、试剂消耗少,污染少和自动化程度高等优点,磁免疫荧光技术具有检测效率高、信号强等...     

Counting of Escherichia coli by a microflow cytometer based on a photonic–microfluidic integrated device

Counting of Escherichia coli DH5α‐cell suspensions in PBS is performed using a microflow cytometer based on a photonic–microfluidic integrated device. Side‐scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a hemocytometer. The detection efficiency is correlated to the ratio of sample to sheath flow rates. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2–4 μm) as their scattering intensities are different.     

基于LIBS的GdFe合金材料快速检测技术研究

为了明晰LIBS（激光诱导击穿光谱）技术检测稀土合金材料的光谱特点,进一步开发基于LIBS的快速检测方法,推动LIBS技术在稀土领域的应用。本文利用激光诱导击穿光谱仪对GdFe合金材料的激发光谱,结合信号强度及背景噪音,研究确定了最佳设置参数。分别考察激光器和光谱仪的参数设置对光谱的影响,根据激发光谱中相关谱线的变化趋势,探讨GdFe合金材料的LIBS检测方法。实验发现,GdFe合金样品与激光孔(Laser aperture)的距离(Z值)、激光能量和积分时间是影响 LIBS光谱信号强度和背景噪音的关键参数。因金属和合金样品表面常覆盖氧化层,考察了脉冲激光的预剥蚀次数对光谱稳定性的影响,研究得出,在双脉冲均为164 mJ激光能量的激发下,预剥蚀1次可保证光谱效果。同时研究了两个激光器同时激发和延迟激发对GdFe合金光谱成因的影响,发现单脉冲激发与双脉冲同时激发所得光谱的信号强度并非两束脉冲单独激发的累加信号。通过光谱分析,确定了Z值0.8 mm、两个激光器的脉冲能量164 mJ、积分时间12 μs、脉冲延时时间0 μs,GdFe合金材料的光谱效果最佳,可为GdFe合金材料的LIBS检测方法建立提供参考,拓展LIBS技术在稀土领域的应用,为稀土合金产品的快速检测技术发展提供基础研究数据。     

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.     

基于表面增强拉曼光谱快速检测纺织品禁用染料

基于表面增强拉曼光谱(SERS)技术, 发展了一种纺织品中染料定性检测的快速方法. 以国家明确禁止使用的致癌染料碱性红9(Basic red 9)和分散黄23(Disperse yellow 23)为模型分子, 利用一步法快速制备的银纳米粒子为SERS基底并进行优化. 通过在纺织品表面直接滴加银纳米粒子的方法实现了纺织品中染料的快速SERS鉴别. 研究结果表明, 该方法不需要复杂的样品前处理过程, 能够直接实现纺织品中染料的快速定性, 且灵敏度高, 对纺织品上两种禁用染料碱性红9和分散黄23的检测限分别为0.16和0.24 mg/kg, 超出了国家标准的要求, 有望成为一种实用的纺织品安全性评估技术.     

基于碳基磁性材料的磁固相萃取技术在食品分析应用中的研究进展

食品中残留的痕量有毒物质严重威胁人体健康,对其进行分析十分必要。然而,食品中有毒物质种类多、量少、基质复杂,需选择适当的样品预处理技术进行提取和净化。磁固相萃取(MSPE)因具有操作简单、省时快速、无需离心过滤、环境友好等优点,被认为是一种高效的样品预处理技术并应用于食品分析中。MSPE中使用的磁性吸附剂的吸附容量和选择性是影响MSPE萃取效率和选择性高低的关键,对所建立分析方法的准确度起着关键作用。碳基磁性材料是具有价格低廉、来源丰富、比表面积大、化学稳定性好、吸附容量高、绿色环保等优点的一类新型功能性磁性材料,可以富集不同性质的有机、无机分析物,在环境分析、生物检测、污染治理等多个领域取得了较大进展。近年来,基于碳基磁性材料的MSPE技术在食品分析预处理领域逐渐得到应用,但尚处于起步阶段,存在巨大的应用潜力。该文以碳基类别(碳纳米管、石墨烯、金属有机骨架衍生碳、活性炭等)为主线,综述了采用MSPE技术,以碳基磁性材料为吸附剂,对食品样品中酯类、真菌毒素、多环芳烃、抗生素、生物碱、酚类、维生素、抗菌药等物质进行萃取,进而采用液相色谱法等进行分析的应用实例,同时阐述了该技术存在的问题,并对其发展方向做出了展望。该综述将为基于碳基磁性材料的MSPE技术在食品分析中的广泛应用提供理论依据和技术支撑。     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

Rapid detection of organophosphorus pesticide residue on Prussian blue modified dual-channel screen-printed electrodes combing with portable potentiostat

A new method consisted of a dual-channel screen-printed electrode (DSPE) efficient modified with Prussian blue and acetylcholinesterase was developed for the rapid detection of organophosphorus pesticide residues.     

Potentiometric detection of polyions based on functionalized magnetic nanoparticles

A novel potentiometric detection strategy based on functionalized magnetic nanoparticles has been developed for rapid and sensitive sensing of polyions. Highly dispersed magnetic nanoparticles coated with ion exchanger and plasticizer could promote an in situ cooperative ion-pairing interaction between the ion exchanger and the polyion analyte in sample solution by dramatically reducing the mass-transfer distance. With applying a magnetic field, the nanoparticles can be attached to the surface of ion exchanger free polymeric membrane. The observed potential signals are related to the polyion concentrations. The proposed polymeric membrane electrode exhibits a linear relationship between the greatest potential response slope (dE/dt) and the logarithm of protamine concentration in the range of 0.05−5 μg/mL with a lower detection limit of 0.033 μg/mL.     

孔雀石绿的磁性免疫层析快速检测方法研究

开发了一种适用于现场快速检测孔雀石绿(MG)的免疫层析试纸条,在超顺磁性纳米微球上偶联MG单克隆抗体作为检测探针,分别将孔雀石绿完全抗原(MG-B SA)和羊抗鼠IgG喷涂于NC膜的T线和C线.结果 发现,T线最佳喷涂量为0.25 mg/mL,抗体最佳偶联量为20 μg,构建的试纸条可在25 min内实现养殖用水及鱼肉...     

Fabrication of Tyrosinase Biosensor Based on Multiwalled Carbon Nanotubes‐Chitosan Composite and Its Application to Rapid Determination of Coliforms

A tyrosinase (Tyr) biosensor was fabricated by immobilizing Tyr on the surface of multiwalled carbon nanotubes (MWNTs)‐chitosan (Chit) composite modified glassy carbon electrode (GCE). The MWNTs‐Chit composite film provided a biocompatible platform for the Tyr to retain the bioactivity and the MWNTs possessed excellent inherent conductivity to enhance the electron transfer rate. The Tyr/MWNTs‐Chit/GCE biosensor showed high sensitivity (412 mA/M), broad linear response (1.0×10^?8–2.8×10^?5 M), low detection limit (5.0 nM) and good stability (remained 93% after 10 days) for determination of phenol. The biosensor was further applied to rapid detection of the coliforms, represented by Escherichia coli (E. coli) in this work. The current responses were proportional to the quantity of coliforms in the range of 10⁴–10⁶ cfu/mL. After 5.0 h of incubation, E. coli could be detected as low as 10 cfu/mL.     

Dual-signal immuno-competitive determination of brain natriuretic peptide based on magnetic nanozyme

Brain natriuretic peptide (BNP) has been a disease marker in the diagnosis of heart failure. In this study, gold nanoparticles modified with Hemin (H-AuNPs) as nanozymes were used to oxidize ABST and MB to amplified colorimetric and electrochemical redox signals respectively. BNP was combined with H-AuNPs (BNP-H-AuNPs) through electrostatic adsorption to construct competitive nanozyme probes. Target BNP in the sample compete with BNP-H-AuNPs to bind the antibody-modified magnetic nanoparticles (AntiBNP-MNPs). Due to the excellent catalytic performance of the nanozyme, BNP can be observed well by colorimetric and electrochemical assays. Electrochemical method ensured more accurate detection of BNP with a wide detection range (1–200 pg/mL) and a low detection of limit (0.03 pg/mL). Meanwhile, the results of the experiment can be easily observed with the naked eye by simple colorimetric method with a range from 5 ng/mL to 25 ng/mL and a limit of detection down to 80.3 pg/mL. Thus, based on the important role of H-AuNPs, this assay has exhibited potential value of detection the other small proteins through this competitive nanozyme method.     

基于PAA——PAN嵌段共聚物胶束制备磁性碳纳米粒子

本文采用原子转移自由基聚合方法合成了聚丙烯酸叔丁酯-聚丙烯腈嵌段共聚物(PtBA-b-PAN), 酸解得到聚丙烯酸-聚丙烯腈两亲嵌段共聚物(PAA-b-PAN). 随后, PAA-b-PAN嵌段共聚物在水溶液中自组装形成以PAA为壳, PAN为核的胶束. 用此胶束为模板, 加入FeCl₃溶液后得到了壳层负载Fe³⁺的聚合物纳米粒子, 经230 ℃空气中预氧化, 600 ℃氮气氛煅烧, 得到了核壳结构的, 具有磁性的碳纳米粒子. 用1H NMR, IR, GPC, TGA, TEM, XRD, AGM等技术对嵌段共聚物及纳米粒子进行了表征, 结果表明纳米粒子的壳层含γ-Fe₂O₃, Fe_2.5C混合物, 核含碳, 直径为35 ± 5 nm, 饱和磁化强度为2.16 emu/g. 在分离、吸波和传感器等方面具有潜在的应用前景.     

Fluorescent detection of protein kinase based on zirconium ions-immobilized magnetic nanoparticles

We report here an affinity separation-based fluorometric method for monitoring the activity and inhibition of protein kinase. In this assay, when the fluorescein isothiocyanate (FITC) labeled substrate peptides (S-peptide) are phosphorylated by kinase, the product peptides (P-peptide) will be adsorbed and concentrated onto the surface of Zr⁴⁺-immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Zr-NTA MNPs) through the chelation of Zr⁴⁺ and phosphate groups. After magnetic separation, the fluorescence intensity of the homogeneous solution changes dramatically. Hence the fluorescence response allows this MNPs-based method to easily probe kinase activity by a spectrometer. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.5 mU μL⁻¹). Moreover, the system is successfully applied to estimate the IC₅₀ value of PKA inhibitor H-89 and detect the Forskolin/3-isobutyl-1-methylxanthine (IBMX) stimulated activation of PKA in cell lysate. Additionally, Zr-NTA MNPs are reusable by stripping Zr⁴⁺ ions from NTA-coated MNPs and rechelating again. This method, which relies on the surface-functionalized MNPs, presents a promising candidate for simple and cost-effective assay of kinase activity and inhibitor screening.