基于背景荧光猝灭-免疫层析法黄曲霉毒素B_1检测卡的研制

基于背景荧光猝灭-免疫层析技术研制快速检测食用油中黄曲霉毒素B1的定量检测卡。依据黄曲霉毒素B1抗原和抗体的活性,筛选检测系统中最佳的黄曲霉毒素B1抗原浓度和抗体标记浓度,应用最佳检测系统进行食用油中黄曲霉毒素B1检测的方法学验证和样品测定。最佳检测系统中黄曲霉毒素B1抗原浓度为0.5 mg/m L,黄曲霉毒素B1抗体标记浓度为1.0μg/m L;用于测定食用油中黄曲霉毒素B1的浓度范围为1.3~50.0 ng/m L,RSD均值为0.42%,平均加标回收率为96.3%~103.2%,RSD均值为2.8%(n=9)。使用有样品采用本法与国标方法的检测结果无显著性差异(p0.05)。本方法专属性强,灵敏度高,可快速、准确的检测食用油中黄曲霉毒素B1。     

背景荧光猝灭-免疫层析法检测C-反应蛋白

采用背景荧光猝灭-免疫层析法(b FQICA),基于双抗体夹心法原理,将合适浓度的捕获抗体、示踪抗体分别固定在试纸条和微孔内,层析时与样本形成抗体-抗原-抗体的夹心结构,建立了一种操作简便、灵敏度高、抗干扰性强且能快速定量检测C-反应蛋白(CRP)的方法.结果表明,CRP在0.0~100.0 ng/m L浓度范围内与相对荧光强度值(F1/F2)呈现良好的相关性,最低检出限为0.0939 ng/m L,加标回收率为87.69%~111.0%,检测3批试剂的批间和批内相对标准偏差均小于15%,与降钙素原(PCT,20.0 ng/m L)及人血清淀粉样蛋白A(SAA,10.0μg/m L)均无交叉反应.采用该方法与免疫透射比浊法同时测定41例临床血清样本,检测结果相关性良好(r=0.9585,P0.01),2种方法无显著性差异(P0.05).     

基于纳米铂-单壁碳纳米管-壳聚糖的癌胚抗原电化学阻抗型免疫传感研究

将单壁碳纳米管-壳聚糖的复合材料(SWCNTs-CS)滴涂在玻碳电极后,采用恒电位沉积的方法在SWCNTs-CS上生成纳米铂,形成Pt NPs-SWCNTsCS复合物后用以固定癌胚抗原抗体(anti-CEA)制成免疫传感界面,在癌胚抗原(CEA)的存在下,在电极表面形成抗原-抗体免疫复合物,以[Fe(CN)6]3-/4-作为混合探针电解质,构建一种简单灵敏无标记的阻抗型免疫分析方法。随着固定CEA量增加,传感器上传递电子的能力下降导致阻抗值增大,且CEA浓度在0.010~12 ng/m L范围时,与阻抗值呈现出良好的线性关系,线性方程为△Retohm=(831.3±17.8)cCEA(ng/m L)+(885.8±95.1)(R2=0.997),检测限为4.2 pg/m L(S/N=3)。该免疫传感器具有良好的稳定性和选择性,回收率在95.0%~108.6%,可初步用于临床对CEA的检测。     

谷胱甘肽稳定的高性能CdTe/CdS核壳型量子点制备及应用于荧光免疫检测米醋中痕量黄曲霉毒素B1

谷胱甘肽作稳定剂水相合成CdTe/CdS核壳型量子点,以EDC/NHS为活化剂对黄曲霉毒素B1(AFB1)抗体进行量子点标记,然后用牛血清蛋白封闭抗体。通过对量子点和标记抗体性能的研究发现,CdTe/CdS核壳型量子点荧光的强度和稳定性较裸壳的CdTe量子点分别提高了4倍和2倍以上。由于谷胱甘肽碳链较长,量子点对抗体尤其是活性位点处的空间构型影响减少,从而改善了量子点标记抗体的稳定性和活性,CdTe/CdS标记的AFB1抗体与AFB1免疫前后荧光强度变化显示抗体至少可以稳定6 d。基于谷胱甘肽稳定的高性能CdTe/CdS量子点,建立了一种荧光免疫检测黄曲霉毒素B1的新方法。AFB1浓度在0.68～40 pmol/L之间荧光强度与浓度呈线性关系,相关系数(R2)为0.9914,检出限为0.3 pmol/L。方法已成功应用于米醋样品中痕量黄曲霉毒素B1的测定。     

基于分子发夹/荧光微球的侧向层析法定量检测黄曲霉毒素B1

基于分子发夹/荧光微球探针构建了一种侧向层析定量检测黄曲霉毒素B1(AFB1)的新方法。一条含有AFB1适配体的分子发夹与荧光微球偶联后,形成的标记探针被喷涂在试纸条的结合垫上。一条5'端含有链霉亲和素的寡核苷酸序列包被在硝酸纤维素膜的检测线上,另一条包含有AFB1适配体互补链的寡核苷酸序列包被在硝酸纤维素膜的质控线上。当待测样本中含有AFB1时,AFB1先与标记物结合垫处标记探针中的AFB1适配体结合,同时,标记探针中的DNA分子发夹‘茎’的双链被打开,AFB1与标记探针形成的复合物层析到反应膜的检测区时,被检测区的寡核苷酸序列捕获,检测区出现亮线。利用该原理,通过一个"off-on"的光信号,实现了对AFB1的高灵敏检测。实验结果表明,AFB1在0.1~50μg/L质量浓度范围内,检测线处的荧光强度(T)和质控线荧光强度(C)的比值与AFB1质量浓度呈良好的线性关系,检出限(S/N=3)达0.05μg/L,且具有很高特异性,可实现对实际样品中AFB1的准确检测。     

快速检测中药材中黄曲霉毒素B1的电化学生物传感器

结合新型纳米传感膜材料,以黄曲霉毒素B1(AFB1)单克隆抗体为生物识别元件,通过考察AFB1与抗体之间的相互作用对测试底液中[Fe(CN)₆]^3-/[Fe(CN)₆]^4-([Fe(CN)₆]^3-/4-)氧化还原体系的影响,构建了一种可用于中药材中AFB1快速检测的电化学生物传感器。在最佳条件下,对AFB1浓度的线性响应范围为0.1~100 pmol/L,检出限为14.7 fmol/L。应用该方法测定大枣、薏苡仁、决明子中AFB1的含量,加标回收率在95.3%~109.5%之间。     

基于金纳米簇荧光“开启”的黄曲霉毒素B_1快速检测方法研究

采用牛血清蛋白作为还原剂和保护剂制备了的金纳米簇,基于Au~+-Hg~(2+)之间的相互作用所导致的金纳米簇荧光淬灭以及Hg2+与黄曲霉毒素B_1(AFB_1)之间的络合作用所导致的荧光恢复,建立了一种快速灵敏的检测AFB_1的方法。通过对该孵育条件的优化,AFB_1的检测线性范围为0.1~10 ng/m L,检测限为0.1 ng/m L。将其应用于实际大米样品中AFB_1的检测,在添加质量浓度为2,5和10 ng/m L时,其回收率在94%~118%之间,相对标准偏差在4.3%~6.3%之间。     

基于纳米银的检测黄曲霉毒素免疫传感器研究

黄曲霉毒素B1(AFB1)是食品中常见的强毒性霉菌毒素,如何对AFB1进行快速、准确、方便的检测受到了研究人员的广泛关注。本研究采用氧化石墨烯/纳米银/AFB1单克隆抗体复合材料(GO/Ag/AFB1抗体)修饰的金电极作为工作电极,利用纳米银良好的导电性实现较低的AFB1检出限。该免疫传感器在pH为7、温度为40℃、抗体与PBS体积比为1∶6000的最优条件下,无需添加信号放大物质,检出限最低可达到7.19×10~(-10 )μg·kg~(-1),检测范围为1.60×10~(-9)～1.68×10~(-5)μg·kg~(-1),孵育时间为30s,回归方程为y=1.3101ln(x)+30.817(R~2=0.9916),测得含有AFB1实际玉米样品的回收率为91.42～107.00%。结果表明,GO/Ag/AFB1抗体免疫传感器具有在无需添加信号放大物质的情况下操作简便、响应迅速、灵敏度高、检出限低等特点,有望应用于食品领域AFB1的实时检测。     

碲化镉纳米晶标记抗荧蒽抗体及其荧光免疫分析

以水热法合成的碲化镉(CdTe)纳米晶标记抗荧蒽抗体后,标记复合物的荧光强度增强,其分散性和稳定性良好。将此标记物用于直接竞争荧光免疫分析,测定了环境水样中荧蒽的含量。结果表明,在0.1～1000μg/L范围内有良好的线性关系;抑制率IC50为12.4μg/L,检出限IC20为13.1ng/L。对水样进行加标回收实验,回收率在95.1%～111%之间;相对标准偏差小于9%。本方法准确可靠,结果满意,能够满足环境中微量环境激素类污染物的检测需要。     

微流控芯片中磁免疫荧光法检测新城疫病毒

建立了一种基于微流控芯片的磁免疫荧光方法,并用于新城疫病毒(NDV)的高灵敏检测。该方法基于抗原抗体免疫识别作用,采用免疫磁珠实现了对NDV的高效捕获和分离,并利用量子点的荧光作为检出信号,提高方法的灵敏度。在优化条件下,NDV的检测限为1ng/mL,线性范围为1～10ng/mL。利用免疫磁珠捕获分离病毒,可用于家禽肝脏、肺、粪便等实际样品中病毒的检测。该方法可以在1h内完成对NDV的检测,操作简单、特异性强,灵敏度高,重现性好。本文为NDV的检测提供了一种快速、准确的方法。     

Rapid detection of aflatoxin B1 on membrane by dot-immunogold filtration assay

Immunofiltration assay for mycotoxins in which nitrocellulose membrane (NCM) was used as a support and enzyme was used as the label has been developed since the late 1980s. As colloidal gold is a good labeling substance that can accelerate antibody-antigen reaction which result can be read directly by naked eyes, the colloidal gold particles could replace the enzyme to be labeled to antibody in aflatoxin B₁ (AFB₁) immunoassay. Dot-immunogold filtration assay (DIGFA) of AFB₁ on NCM was developed in this study. At first, the colloidal gold was synthesized and colloidal gold-monoclonal antibody (McAb) conjugates against AFB₁ were prepared at pH 7.0 of colloidal gold solution, 0.018 mg/mL of McAb. Then the colloidal gold-McAb conjugates were used to develop AFB₁ DIGFA, which detection time was only 15 min, six times less than that of ELISA. With this method to determine the standard AFB₁ solution, the results demonstrated a visual detection limit of approximately 2 ng/mL of AFB₁, which was similar to that of ELISA. This method had good specificities for AFG₁, AFG₂ and AFM₁ and a little cross-reactivity with AFB₂. 45 food samples collected from the markets were subjected to DIGFA and the results showed that one corn sample was positive and in agreement that of HPLC. It is suggested that DIGFA developed in current study has a potential use as a rapid and cost-effective screening tool for the determination of AFB₁ in foods in the field within 15 min without complicated steps.     

Modelling of the impedimetric responses of an aflatoxin B1 immunosensor prepared on an electrosynthetic polyaniline platform

Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. An electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) was developed with AFB₁antibody (AFB₁-Ab) immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of the Pt/PANi–PSSA electrode, the Pt/PANi–PSSA/AFB₁-Ab immunosensor and Pt/PANi–PSSA/AFB₁-Ab incubated in bovine serum albumin (BSA) were 0.458, 720 and 1,066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for the Pt/PANi–PSSA electrode to 1,066 kΩ for the Pt/PANi–PSSA/AFB₁-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB₁ immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩ L/mg.     

Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples

A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B₁ (AFB₁), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB₁ concentrations determinable by ELISA ranged from 0.1 to 10 μg L⁻¹. The IC₅₀ value was 0.62 μg L⁻¹. Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP–AFB₁ conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP–AFB₁ conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L⁻¹ calcium chloride in 0.05 mol L⁻¹ Tris-HCl buffer (pH 7.2) maintained the activity of HRP–AFB₁ at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4°C, whereas the untreated MAb-coated plate lost its activity within 2 days.     

Novel Enzyme-Linked Aptamer Assay for the Determination of Aflatoxin B1 in Peanuts

Abstract

A novel enzyme-linked aptamer assay is reported for the determination of aflatoxin B₁ (AFB₁). AFB₁ can competitively bind with the immobilized biotin-aptamer and release biotin complementary DNA, leading to the gradual fading of the detection system color with increasing of AFB₁ concentration. In the absence of AFB₁, the biotinylated complementary DNA is not be released from the fixed aptamer. Therefore, the enzyme reaction occurs in the detection system. Under the optimized experimental conditions, the proposed method possessed a wide linear range for AFB₁ from 1 to 80?ng/mL (R² of 0.990) with a low detection limit of 0.36?ng/mL. The method was then applied to detect uncontaminated peanuts fortified with different concentrations of AFB₁. The recovery values were from 82.60% to 94.43%, which indicated the proposed method may be used to detect AFB₁ in food and has potential for the development of test kits.     

A sensitive and highly stable electrochemical impedance immunosensor based on the formation of silica gel-ionic liquid biocompatible film on the glassy carbon electrode for the determination of aflatoxin B1 in bee pollen

The paper describes a sensitive and highly stable label-free electrochemical impedance immunosensor for the determination of aflatoxin B₁ (AFB₁), which is based on the formation of silica gel-ionic liquid biocompatible film on the glassy carbon electrode. The electrochemical performances of the sensor were investigated by electrochemical impedance spectroscopy using a Fe(CN)₆^3−/4− phosphate buffer solution as base solution for test. As new ionic liquid, 1-amyl-2,3-dimethylimidazolium hexafluorophosphate, offers a very biocompatible microenvironment for AFB₁ antibody, the sensor exhibits good repeatability (RSD = 1.2%), sensitive electrochemical impedance response to AFB₁ in the range of 0.1-10 ng ml⁻¹ and lowers the detection limit of AFB₁ (0.01 ng ml⁻¹)_. The electron-transfer resistance change of the sensor after and before incubation with AFB₁ of 2.0 ng ml⁻¹ can retain 95% over a 180-day storage period at 4 °C. The results present a remarkable improvement of sensitivity (2-fold) and long-term stability (190-fold) when compared to classical silica gel sensor. Moreover, proposed sensor has a high selectivity to AFB₁ alone with no significant response to AFB₂, AFG₁, AFG₂ and AFM₁ as single substrates, it has been successfully applied to the determination of trace AFB₁ in bee pollen samples with a spiked recovery in the range of 96.0-102.5%.     

On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA_1c, which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA_1c in blood calibrator samples from other Hbs, e.g. HbA₀, HbA_1a or HbA_1b, were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA_1a and HbA_1b, HbA_1c and HbA₀ fractions were eluted in this order. A linear relationship between HbA_1c levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA_1c. All procedure including antigen-antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA_1c.     

Safety evaluation of Semen Sojae Preparatum based on simultaneous LC–ESI–MS/MS quantification of aflatoxin B1, B2, G1, G2 and M1

Semen Sojae Preparatum (SSP) is one of the most widely used traditional Chinese medicines, and is also a functional food. However, contamination with aflatoxins may occur in the fermentation process. To evaluate its safety, an accurate and rapid LC–ESI–MS/MS analytical method was developed and validated for the simultaneous determination of AFB₁, AFB₂, AFG₁, AFG₂ and AFM₁ in SSP. After a simple ultrasonic extraction of SSP samples, chromatographic separation was achieved on an Agilent Zorbax SB‐C₁₈ column (2.1 × 50 mm, 3.5 μm) with a flow rate of 0.50 mL/min. The gradient elution program was performed using a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. Detection of five aflatoxins was based on triple quadrupole mass spectrometry using a multiple reaction monitoring mode with an electrospray ionization source. SSP is likely to be contaminated by aflatoxins in the processes of fermentation, storage, transportation and usage, and it is necessary to strictly monitor it. Artemisia annua L. and Morus alba L. may inhibit the production and growth of AFB₁‐ and AFB₂‐producing fungi, which has a certain detoxification effect on contamination with aflatoxins in SSP.     

AFB1–AP Conjugate for Enzyme Immunoassay of Aflatoxin B1 in Corn Samples

Abstract

This article describes the conjugation between aflatoxin B₁ (AFB₁), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B₁ determination in corn is presented. Three AFB₁–AP conjugates in different toxin–enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%.     

Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples

Membrane-based immunoassay has been developed for simultaneous estimation of aflatoxin B₁ (AFB₁) and ochratoxin A (OA) in chili samples. The combined estimation of both the mycotoxins is more economical in respect of time, work and materials than two separate assays. The method uses a low cost test device consisting of a membrane with immobilized anti-AFB₁ and anti-OA antibodies and a filter paper attached to a polyethylene card below the membrane. It allows direct analysis of sample extracts containing substantial amount (40%) of methanol. This permits the use of two-fold diluted sample extracts resulting in minimum dilution error. The limit of quantitation obtained was 2 and 10 μg kg⁻¹ for AFB₁ and OA, respectively. The tolerance of 40% methanol was found to be due to the application of small size (0.8 mm diameter) spots on membranes, as the tolerance decreases to 20% with gradual increase in spot size. The combined method is capable of producing acceptable results to analyze AFB₁ and OA in chili with accuracy and precision. The AFB₁ and OA values obtained for spiked and naturally contaminated chili samples by the simultaneous method were in good correlation with those measured by individual ELISA. The method offers a simple, rapid and cost-effective screening tool to meet the requirements of the rapidly evolving EU legislation.     

Filtration-based tyramide amplification technique—a new simple approach for rapid detection of aflatoxin B<Subscript>1</Subscript>

The catalyzed reporter deposition (CARD) method of signal amplification, also called “tyramide signal amplification”, has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B₁ (AFB₁) in a variety of foodstuffs with a detection limit of 12.5 μg kg⁻¹. The assay procedure involves sequential addition of standards or sample, AFB₁–horseradish peroxidase (HRP) conjugate, B-T, avidin–HRP, and substrate solution over anti-AFB₁ antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB₁ at concentrations from 25 to 100 mg kg⁻¹ were between 95 and 105%. AFB₁ results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC. Figure The principle of filtration-based tyramide filtration technique