A simple method for the fluorodensitometric determination of aflatoxin M1 in milk powder

A simple and accurate method for the fluorodensitometric determination of aflatoxin M₁ is described. Aflatoxin M₁ is extracted into chloroform and spotted on a silica gel thin-layer chromatography plate. A solvent system of ethyl ether-methanol-water (96:3:1) is used. The spots of aflatoxin M₁ are evaluated by direct densitometric light emission measurements.     

Aflatoxin B1 and M1 in milk

The aflatoxin M₁ (AFLAM₁) is a mycotoxin that results from the hydroxylation of the aflatoxin B₁ (AFLAB₁). It contaminates the milk of animals fed with a diet containing its precursor. In this work, we determined the occurrence of AFLAB₁ and AFLAM₁ in milk, as well as the chromatographic conditions to quantify these mycotoxins. The extraction and quantification of AFLAB₁ and AFLAM₁ in naturally contaminated and artificially spiked milk samples which are produced and marketed in the state of RS were performed using the AOAC official method and UHPLC with fluorescence detection. We obtained a separation factor of 2.3 for AFLAB₁ and AFLAM₁ using a mobile phase consisting of 1% acetic acid:acetonitrile:methanol (55:10:35). The analytical curves had a wide linearity range and the limit of quantification (LOQ_m) concentrations of AFLAB₁ and AFLAM₁ were equal to 0.5 and 0.25 μg L⁻¹, respectively. Samples of pasteurized and ultra-high-temperature processed (UHT) milk showed natural contamination, and the levels for both aflatoxins ranged from 0.7 to 1.5 μg L⁻¹. Raw and concentrated milk samples only contained AFLAM₁, with a maximum average concentration of 1.7 μg L⁻¹. These concentrations, higher than permitted by legislation, confirm the existence of a health risk, as well as highlight the relevance of searching for alternatives to reduce this contamination.     

Direct and ultrasensitive optofluidic-based immunosensing assay of aflatoxin M1 in dairy products using organic solvent extraction

Aflatoxin M₁ (AFM₁), a highly toxic secondary metabolite, is present in a wide range of dairy products. In this study, we designed a simple, low-cost, reusable, and easy-to-operate immunosensing method for ultrasensitive detection of AFM₁ in dairy products by using a portable evanescent wave-based optofluidic biosensing platform (EOBP). The developed method provides the minimum detection limit of 5 ng/L, which is below the most restrictive standard imposed by the current regulations for AFM₁ in dairy products. The effect of several organic solvents, such as methanol, acetone, and acetonitrile, on the binding reaction of antibody-antigen in heterogeneous and homogeneous solutions was evaluated. Although the effect of organic solvents on the homogeneous binding reaction between antibody and antigen is more significant than that of heterogeneous binding reaction between antibody in solution and antigen immobilized onto the sensor surface, the fluorescence signal detected by EOBP is linearly dependent on AFM₁ concentration. Therefore, AFM₁ can be directly quantified even if the samples contain a certain organic solvent concentration. The robustness and stability of AFM₁-ovalbumin conjugate allow the regeneration of modified biosensor surface for more than 200 times, thereby achieving a cost-effective and reliable AFM₁ determination. The proposed method provides a rapid, ultrasensitive, and reliable AFM₁ determination in dairy products without complicated sample pretreatment process.     

Magnetic beads-based multicolor colorimetric immunoassay for ultrasensitive detection of aflatoxin B1

Aflatoxin B₁ (AFB₁) is one of the most toxic, mutagenic and carcinogenic mycotoxin, widely exists in contaminated food, grains and feedstuff products. In this study, a novel magnetic beads multicolor colorimetric immunoassay (MBMCIA) based on Au@Ag nanorods (Au@Ag NRs) is proposed to visual detect ultralow concentration of AFB₁ with high-resolution by the naked-eye. To design the MBMCIA system, AFB₁-BSA conjugates were first coated on the surface of magnetic beads (MBs), then alkaline phosphatase (ALP) as a bridge between immunoassay and color reaction was used for catalytic hydrolysis of ascorbic acid-phosphate to generate reductive ascorbic acid. Finally, the yielded ascorbic acid could reduce silver ions to grow a silver coating on the surface of gold nanorods to generate Au@Ag NRs, which leads to the bule-shifted longitudinal absorption peak of Au NRs, accompanying with a series of perceptible color change. Under the optimal conditions, the proposed MBMCIA exhibited good sensitivity and specificity for the detection of AFB₁ with the detection limit as low as 5.7 pg/mL. Meanwhile, the MBMCIA was also applied for the analysis of AFB₁ in spiked wheat samples, the obtained recoveries range from 99.1% to 104.3% with relative standard deviation (RSD) less than 7.05% were acceptable. The proposed MBMCIA integrates separated, enriched, anti-interference and signal read-out into one, which opens up a new avenue for an on-site visual food safety inspection or environmental monitoring.     

Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M₁ (AFM₁) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM₁ surrogate, was generated by immunizing rabbits with F(ab′)₂ fragments from the anti-AFM₁ monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM₁ mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM₁ and the anti-Id antibody (y = 31.91x − 8.47, r = 0.9997). The assay was applied to analyze AFM₁ in spiked milk samples. The IC₅₀ value of the surrogate calibrator curve was 2.4 μg mL⁻¹, and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y = 0.81x + 9.82, r = 0.9922).     

Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL^?1 in buffer and 0.11 μg L^?1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

Ultrasonic Synthesis of Gold Nanoparticles and Its Use in Immunochromatographic Assay for Detection of Kanamycin

An ultrasonic method to synthesize gold nanoparticles with uniform size was reported. Effects of ethanol concentration, solution pH, ultrasonic irradiation power, and time on the formation of gold nanoparticles were investigated. The obtained gold nanoparticles were characterized by ultraviolet-visible (UV-vis) absorption spectra and scanning electron microscope (SEM). The method performed in ethanol solution under ultrasonic irradiation is friendly to the environment. Furthermore, a nanogold-labeled probe was used to develop an immunochromatographic method for detection of kanamycin.     

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL⁻¹) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL⁻¹ its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).     

超高效液相色谱-串联质谱法测定牛奶中黄曲霉毒素M1

提出了超高效液相色谱-串联质谱法测定牛奶中黄曲霉毒素M₁含量的方法。样品经免疫亲和柱净化,用乙腈洗脱下来。以ACQUITY UPLC BEH C₁₈柱（2.1 mm×50mm,1.7μm）为分离柱,乙腈和水为流动相梯度淋洗,用串联质谱测定。采用电喷雾电离正离子模式进行多反应监测。黄曲霉毒素M₁的质量浓度在0.1～16.0μg·L^-1范围内与其峰面积呈线性关系,检出限（3S/N）为0.001 5μg·kg^-1,测定下限（10S/N）为0.005μg·kg^-1。方法用于分析牛奶样品,测得回收率在89.5%～101.5%之间,测定值的相对标准偏差（n=6）在2.8%～9.5%之间。     

Surface properties of new virginiamycin M1 derivatives

Three kinds of derivatives of the M₁ factor of virginiamycin have been synthesised: esters with long chain fatty acids, oximes with modified polar amino acids and bis-derivatives with both the ester and oxime function. The study of the surface tension time dependence of M₁ and its derivatives has shown that it is necessary to enhance simultaneously the hydrophobicity and the hydrophilicity of M₁ to render M₁ surface-active. A structure/function relationship study of the surface-active bis-derivatives has shown that enhancing the hydrophobicity of the molecule led to slower adsorption kinetics, higher stability of the monolayers formed and a better capacity to penetrate a membrane model. The repulsive electrostatic forces due to the presence of charges on the amino acids linked to M₁ lead to higher surface tensions, a greater molecular area at the interface and lower penetration into a membrane model.This study has demonstrated that modifying systematically the hydrophobicity and hydrophilicity of a non surface-active molecule allows the production of surface-active derivatives.     

Limitations in the use of horseradish peroxidase as an enyzme probe in the development of a homogeneous immunoassay for aflatoxin B1

Horseradish peroxidase (HRPO) was used as a probe to quantitate aflatoxin B₁ by a homogeneous immunoassay. The conjugation of AFB₁ to HRPO resulted in 54% loss of enyzme activity. In the presence of AFB₁ specific antibodies, the HRPO-AFB₁ conjugate showed reversal of its lost enzyme activity by 12%. This positive modulatory effect of antibody on the enzyme activity was used as an analytical tool to quantitate AFB₁. The homogeneous assay carried out with free AFB₁ and HRPO-AFB₁ conjugate in the presence of antibodies indicated poor linearity as compared to the heterogeneous assay. It was observed that the number of HRPO-lysine residues involved in AFB₁ conjugation were 6–8. The low level of modulation of enzyme activity by antibody with respect to HRPO-AFB₁ conjugate, could possibly be attributed to the limited number of lysine residues in the HRPO molecule and its proximity to the active site of the enzyme. Thus, HRPO was found to be limiting as an enzyme with respect to the homogeneous enzyme immunoassay for AFB₁ analysis. The antibodies raised were specific for AFB₁, and showed excellent linearity even at high dilution for the detection of AFB₁ by ELISA indicating that antibodies per se were not the limiting factor.     

Magnetically assisted solid phase extraction using Fe3O4 nanoparticles combined with enhanced spectrofluorimetric detection for aflatoxin M1 determination in milk samples

A novel, facile and inexpensive solid phase extraction (SPE) method using ethylene glycol bis-mercaptoacetate modified 3-(trimethoxysilyl)-1-propanethiol grafted Fe₃O₄ nanoparticles coupled with spectrofluorimetric detection was proposed for determination of aflatoxin M1 (AFM1) in liquid milk samples. The method uses the advantage fluorescence enhancement by β-cyclodexterin complexation of AFM1 in 12% (v/v) acetonitrile–water and the remarkable properties of Fe₃O₄ nanoparticles namely high surface area and strong magnetization were utilized to achieve high enrichment factor (57) and satisfactory extraction recoveries (91–102%) using only 100 mg of magnetic adsorbent. Furthermore, fast separation time of about 15 min avoids many time-consuming column-passing procedures of conventional SPE. The main factors affecting extraction efficiency including pH value, desorption conditions, extraction/desorption time, sample volume, and adsorbent amount were evaluated and optimized. Under the optimal conditions, a wide linear range of 0.04–8 ng mL⁻¹ with a low detection limit of 0.015 ng mL⁻¹ was obtained. The developed method was applied for extraction and preconcentration of AFM1 in three commercially available milk samples and the results were compared with the official AOAC method.     

Liposomes loaded with quantum dots for ultrasensitive on-site determination of aflatoxin M1 in milk products

A quantitative fluorescence-labeled immunosorbent assay and qualitative on-site column tests were developed for the determination of aflatoxin M1 in milk products. The use of liposomes loaded with quantum dots as a label significantly increased the assay sensitivity by encapsulating multiple quantum dots in a single liposome and, therefore, amplifying the analytical signal. Two different techniques were compared to obtain aflatoxin–protein conjugates, used for further coupling with the liposomes. The influence of nonspecific interactions of the liposome-labeled conjugates obtained with the surface of microtiter plates and column cartridges was evaluated and discussed. The limit of detection for fluorescence-labeled immunosorbent assay was 0.014 μg kg^-1. For qualitative on-site tests, the cutoff was set at 0.05μg kg^-1, taking into account the EU maximum level for aflatoxin M1 in raw milk, heat-treated milk, and milk for the manufacture of milk-based products. The direct addition of labeled conjugate to the milk samples resulted in an additional decrease of analysis time. An intralaboratory validation was performed with sterilized milk and cream samples artificially spiked with aflatoxin M1 at concentrations less than, equal to and greater than the cutoff level. It is shown that milk products can be analyzed without any sample preparation, just diluted with the buffer. The rates for false-positive and false-negative results were below 5 % (2.6 % and 3.3 %, respectively).

A competitive immunoassay for ultrasensitive detection of Hg in water,human serum and urine samples using immunochromatographic test based on surface-enhanced Raman scattering

An immunochromatographic test (ICT) strip was developed for ultrasensitive competitive immunoassay of Hg²⁺. This strategy was achieved by combining the easy-operation and rapidity of ICT with the high sensitivity of surface-enhanced Raman scattering (SERS). Monoclonal antibody (mAb) against Hg²⁺ and Raman active substance 4-mercaptobenzoic acid (MBA) dual labelled gold nanoparticles (GNPs) were prepared as an immunoprobe. The Raman scattering intensity of MBA on the test line of the ICT strip was measured for quantitative determination of Hg²⁺. The ICT was able to directly detect Hg²⁺ without complexing due to the specific recognition of the mAb with Hg²⁺. The IC₅₀ and limit of detection (LOD) of the assay for Hg²⁺ detection were 0.12 ng mL⁻¹ and 0.45 pg mL⁻¹, respectively. There was no cross-reactivity (CR) of the assay with other nineteen ions and the ICT strips could be kept for 5 weeks without loss of activity. The recoveries of the assay for water, human serum and urine samples spiked with Hg²⁺ were in range of 88.3–107.3% with the relative standard deviations (RSD) of 1.5–9.5% (n = 3). The proposed ICT was used for the detection of Hg²⁺ in urine samples collected from Occupational Disease Hospital and the results were confirmed by cold-vapor atomic fluorescence spectroscopy (CV-AFS). The assay exhibited high sensitivity, selectivity, stability, precision and accuracy, demonstrating a promising method for the detection of trace amount of Hg²⁺ in environmental water samples and biological serum and urine samples.     

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.     

Electrochemical immunosensor for determination of aflatoxin B1 in barley

This paper reports the assembly of a disposable electrochemical immunosensor based on the indirect competitive enzyme linked immunosorbent assay (ELISA), for simple and fast measurement of aflatoxin B₁ (AFB₁) in barley using differential pulse voltammetry (DPV). The immunosensor strip was assembled immobilising the biological component (i.e. the AFB₁ conjugated to bovine serum albumin, incubation the sample (or standard) with the monoclonal antibody anti-AFB₁ (MAb). A spectrophotometric ELISA was used in a preliminary phase of development, prior to transferring the assay to the SPEs.

Results showed a detection limit of 20 and 30 pg/mL for the spectrophotometric ELISA and the electrochemical immunosensor, respectively.

The extraction efficiency and the matrix effect have been evaluated by spiking blank barley with AFB₁ before and after the sample treatment. After treatment, samples were analysed using a 1:10 (v/v) dilution in PBS (phosphate-buffered saline, pH 7.4) in order to minimise the matrix effect. Good recoveries were obtained, which demonstrated the suitability of the proposed method for routine screening of AFB₁ in barley.     

Rapid test strips for analysis of mycotoxins in food and feed

An overview is given on recent trends and applications of rapid immunodiagnostic tests for screening of food and feed for mycotoxins. Different test formats are discussed, and challenges in the development of lateral-flow devices for on-site determination of mycotoxins, with requirements such as being robust, fast, and cost-effective, are briefly elucidated.     

Development of a chemiluminescence-based quantitative lateral flow immunoassay for on-field detection of 2,4,6-trinitrotoluene

Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen–antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2 μg mL⁻¹ TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5 μg mL⁻¹ TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion).     

Development of a competitive liposome-based lateral flow assay for the rapid detection of the allergenic peanut protein Ara h1

A competitive lateral flow assay for detecting the major peanut allergen, Ara h1, has been developed. The detector reagents are Ara h1-tagged liposomes, and the capture reagents are anti-Ara h1 polyclonal antibodies. Two types of rabbit polyclonal antibodies were raised either against the entire Ara h1 molecules (anti-Ara h1 Ab) or against an immunodominant epitope on Ara h1 (anti-peptide Ab). All of them reacted specifically with Ara h1 in Western Blot against crude peanut proteins. Moreover, the anti-Ara h1 Ab was chosen for this assay development because of its highest immunoactivity to Ara h1-tagged liposomes in the lateral flow assay. The calculated limit of detection (LOD) of this assay is 0.45 g mL^–1 of Ara h1 with a dynamic range between 0.1 and 10 g mL^–1 of Ara h1 in buffer. Additionally, the visually determined detection range is from 1 to 10 g mL^–1 of Ara h1 in buffer. Results using this assay can be obtained within 30 min without the need of sophisticated equipment or techniques; therefore, this lateral flow assay has the potential to be a cost-effective, fast, simple, and sensitive method for on-site screening of peanut allergens.