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Keywords:

  • hydrophilic imprinted membrane;
  • biomimetic antibody;
  • enzyme-linked immunosorbent assay;
  • acrylamide

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

BACKGROUND

Acrylamide has attracted worldwide concern due to its neurotoxicity, genotoxicity, reproductive–development toxicity. It is necessary to develop an accurate and reliable analytical method to prevent the harm on the human health.

RESULTS

In this study, a sensitive and fast analytical method of direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) was developed using a hydrophilic imprinted membrane as biomimetic antibody. This novel imprinted membrane was directly synthesised on the well surface of MaxiSorp polystyrene 96-well plates in an aqueous environment, and it exhibited high binding ability and specificity toward acrylamide. Under the optimal conditions, the established BELISA method had a good sensitivity (IC50, 8.0 ± 0.4 mg L−1) and a low limit of detection (IC15, 85.0 ± 4.2 µg L−1). The blank potato samples spiked with acrylamide at three levels of 100, 250 and 500 µg L−1 were extracted and determinate by the proposed method, and good recoveries ranging from 90.0% to 110.5% were obtained. This presented method was applied to the quantitative detection of the acrylamide in French fries and cracker samples. Also, the results were correlated well with that obtained by the gas chromatography method.

CONCLUSION

With good properties of high sensitivity, simple pre-treatment and low cost, this BELISA could be a promising screening method in food sample analysis. © 2013 Society of Chemical Industry


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In 2002, the Swedish National Food Administration and researchers of Stockholm University first announced that high levels of acrylamide (2-propenamide) had been found in food products, especially in fried and baked products containing carbohydrates and amino acids.[1-5] Many results indicated that acrylamide is usually produced in starchy foodstuffs during the Maillard reaction process at high temperature (120–180°C). This compound has attracted worldwide concern due to its neurotoxicity, genotoxicity and reproductive–development toxicity,[6-8] and it has been classified as a probable human carcinogen by the International Agency for Research on Cancer.[9] To control acrylamide and prevent harm to human health, the development of a fast and reliable analytical method is of great significance.

A variety of analytical methods for screening acrylamide and acrylamide metabolites has been developed, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography or liquid chromatography coupled with mass spectrometry (GC-MS, LC-MS) and enzyme-linked immunosorbent assay (ELISA).[10-13] Among them, the GS-MS and LC-MS methods are effective and sensitive. However, they have many potential drawbacks, including an investment in expensive instrumentation, extensive clean-up and purification pre-treatment. For GC and GC-MS, a complex derivatisation is usually required prior to analysis. Such costly and time-consuming instrumental techniques lead to the limitation of their wide utilisation and direct field detection. With the merits of rapid analysis, high sensitivity and operating facility, ELISA has become a popular and useful screening tool. In recent years, several ELISA methods for the determination of acrylamide have been developed.[14-16] However, the high commercial cost and difficulties associated with antibody production due to the diminutive size of acrylamide (71.08 Da), together with the need for laboratory animals, are often considered as challenging problems of ELISA. Furthermore, the biological antibodies are relatively unstable, which leads to a limitation of their application and development.

In order to overcome the above shortcomings, much effort has been expended in attempting to design and synthesise antibody-like receptors that could replace biological antibodies. Molecular imprinting technology is one of the promising methods for synthesising functionalised material.[17, 18] The molecularly imprinted polymers (MIPs) thus prepared exhibit high selectivity, physico-chemical stability and applicability in harsh chemical media, and have been applied in separation processes and chemical sensors.[19-26] Use as an artificial antibody in immunoassay-like analysis is one of the most interesting and exciting applications of MIPs, and some MIP-based immunoassay methods have been reported.[27-29] However, most of the working solution of ELISA are aqueous buffers, whereas the traditional preparation methodologies for MIPs are organic polymer-based systems, and their selectivity in aqueous environments is poor. Furthermore, the structure of enzyme-labelled antigen is quite different from the template molecule, and it is difficult for the enzyme conjugates to enter the imprinted cavity and combine with the binding sites. Therefore, the sensitivity of these methodologies is lower and the cross-reactivity is higher than the traditional ELISA method.[30-34]

The objective of this work was to prepare an imprinted membrane on the well surface of 96-well plates with high accessibility and binding ability in aqueous environment. Using it as the antibody, an improved and sensitive direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method was developed. The parameters affecting the performance of this BELISA method are optimised in detail. The applicability and accuracy of this presented method are also evaluated.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Samples

The French fries and crackers were purchased from the supermarket of Tai' an (Shandong, China) in November 2012.

Chemicals and materials

Acrylamide, β-alanine, 8-aminocaprylic acid, acrylic acid, glycinamide hydrochloride and acryloyl chloride were purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Methacrylic acid (MAA), 2,2-azobisisobutyronitrile (AIBN) and anhydrous pyridine were bought from Tianjin Chemical Reagent Factory (Tianjin, China), and they were purified before use. Ethyleneglycol dimethacrylate (EGDMA), 3,3',5,5'-tetramethylbenzidine (TMB), horseradish peroxidase (HRP), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) were supplied by Sigma–Aldrich (St Louis, MO, USA). All the other solvents and reagents used in this study were of the highest available purity and at least of analytical grade. Doubly deionised water (DDW) obtained from a Water Pro water system (Labconco Corp., Kansas City, MO, USA) was used throughout the experiments.

Maxisorp polystyrene 96-well plates (Corning, Costar) were purchased from Beijing Biolead Biology Sci & Tech Co., Ltd (Beijing, China). The thin-layer chromatography (TLC) plates were obtained from Merck (Darmstadt, Germany).

Solutions

The solutions of phosphate-buffered saline (PBS, 50 mmol L−1 sodium phosphate, 154 mmol L−1 NaCl, pH 7.0), PBS/T (PBS with 0.05% Tween-20), substrate solution (1.25 mmol L−1 3,3′,5,5′-tetramethylbenzidine, 1.6 mmol L−1 hydrogen peroxide in acetate buffer, pH 5.0) and stopping solution (1.25 mol L−1 sulfuric acid) were used in this study.

Instruments

A SEI 15kVon a SS40 scanning electron microscope (Shimadzu, Kyoto, Japan) was used in this study. Immunoassay absorbance was read in a dual wavelength mode (450–650 nm) with a Multimode Plate Reader (Molecular Devices, MenloPark, California, USA).

A 2010 gas chromatograph (Shimadzu, Japan) equipped with a flame photometric detector was used for determination of 2-bromopropionamide (derivational product of acrylamide with bromination). The separation was conducted on an RTX-WAX capillary column (30 m × 300 µm i.d. × 0.25 µm film thickness). Nitrogen was used as the carrier gas at the constant flow rate of 1.0 mL min−1, and the injection volume was 1.0 µL. The injection port temperature was held at 225°C at the split mode with the split ratio of 10:1. The detector temperature was held at 250°C. The temperature program was as follows: 110°C held for 1 min, then increased to 180°C at a rate of 20°C min−1 and held for 2 min. After that, the temperature was increased to 190°C at 2.0°C min−1. Finally, the temperature was increased to 240°C at 25°C min−1 and held for 6 min.

Haptens synthesis

The hapten B was synthesised as follows: 0.89 g β-alanine (10 mmol) was dissolved in 3 mol L−1 potassium hydroxide solution within a pH range of 8–9. When 1 mL acryloyl chloride (15 mmol) was added drop-wise, the mixture was stirred at 0°C for 1 h. After reaction for another 4 h at room temperature, the mixed solution was washed with diethyl ether (20 mL × 2). The water phase was collected and adjusted the pH to 3.0 using 5 mmol L−1 HCl, and then extracted with chloroform (20 mL) three times. The organic fractions was dried by MgSO4, filtered and evaporated to dryness under reduced pressure. Finally, the crude product was crystallised with diethyl ether. Rf = 0.33 (ethyl acetate–petroleum ether–chloroform, 3:2:1, v/v/v).

Hapten C was prepared from 8-aminocaprylic acid (1.59 g, 10 mmol) and acryloyl chloride (1 mL, 15 mmol) following above procedure. Rf = 0.50 (ethyl acetate–petroleum ether–chloroform, 3:2:1, v/v/v). Hapten A (acrylic acid) and hapten D (glycinamide hydrochloride) were bought directly. The structures of four haptens are shown in Fig. 1.

image

Figure 1. Chemical structures of acrylamide haptens used in this study.

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Preparation of enzyme conjugates

In this study, the enzyme conjugates A, B and C was prepared by an active ester method respectively, and the enzyme conjugates D was synthesised by the glutaraldehyde method.

The enzyme conjugate A was synthesised as follows: 0.03 mmol NHS (3.4 mg) and 0.06 mmol DDC (12.4 mg) were added to the active ester of hapten (2.16 mg hapten A dissolved in 216 µL DMF), and the mixture was stirred at room temperature for 4 h. After centrifugation, the supernatant was preserved as solution (a); 10 mg horseradish peroxidise (HRP) was added to 2 mL 50 mmol L−1 K2HPO4 as solution (b). Solution (a) was slowly transferred to solution (b) at 4°C drop by drop, and the reaction solution was gently shaken by hand. The mixture was then placed at 4°C overnight. Finally, the enzyme conjugated solution was dialysed against phosphate buffered saline (pH 7.0) for 3 days and stored at 4°C. The enzyme conjugates B and C were prepared according the same method of enzyme conjugates A.

The enzyme conjugates D was synthesised as follows:[16, 35, 36] 2 mL glycinamide hydrochloride (3.4 mg, 0.03 mmol) and HRP (10 mg) were dissolved in Na2HPO3 solution, and the pH was adjusted to 9.3, then 5.7 µL of 25% glutaraldehyde was added drop-wise at 0°C. After reaction for 4 h at room temperature, the mixture was placed at 4°C overnight. After that, the enzyme conjugated solution was dialysed against phosphate-buffered saline (pH 7.0) for 3 days and stored at 4°C before use.

Grafting of an acrylamide-imprinted membrane to the surface of a 96-well plate

The imprinted membrane was directly polymerised on the wells of 96-well plates according to the following procedure: 0.14 g acrylamide (2.0 mmol) and 0.3444 g MAA (4.0 mmol) were dissolved in 8 mL water and 12 mL acetonitrile. After stirring by the magnetic stirrer at room temperature for 60 min, 1.59 mL EGDMA (8.0 mmol) and 20 mg AIBN were added. The mixture was stirred for another 120 min, and then 200 µL of the mixture was placed in each well of a Maxisorp polystyrene 96-well plate. The reaction was carried out for 18.0 h in a plastic bag filled with nitrogen gas at 38°C. When the polymerisation ended, the 96-well plates were washed with deionised water three times to remove the unreacted reagents and template molecules. The imprinted membranes on the 96-well plate were extracted firstly with 300 mL methanol–acetic acid (7:1, v/v) for 8.0 h, then with 300 mL methanol for 4.0 h to be free of acrylamide. Finally, the 96-well plate was dried in a vacuum oven at 38°C for 4.0 h.

For compare, a traditional imprinted membrane was synthesised following above procedure except using 20 mL methanol as the reaction solution. A non-imprinted membrane was also prepared following the same procedure without the addition of acrylamide.

Preparation of standard solutions

For the construction of calibration curve, six standard solutions containing acrylamide in PBS solution in the range of 16.0 µg L−1 to 50.0 mg L−1 were prepared freshly in glass tubes. The stock solution (1000 mg L−1 in DDW) of acrylamide was diluted to 1/20 and the solution of 50 000 µg L−1 (in PBS) was sequentially diluted to 10 000, 2 000, 400, 80 and 16 µg L−1.

Procedure for direct competitive ELISA

The direct competitive ELISA was carried out using the imprinted membrane on the 96-well plate as biomimetic antibody. Plates were washed three times with PBST [PBS with 0.05% (v/v) Tween 20]. In the blank and control wells, 200 µL and 100 µL PBS solution was added, respectively. Gradient standard solutions or sample extracts were applied to the allocated wells (100 µL well−1). Then, 100 µL enzyme conjugate was added immediately to each well except for the blank wells. After that, the 96-well plate was shaking for 1.0 h (200 times min−1) at room temperature by a horizontal shaker. Following the washing with PBST for five times, 150 µL of substrate solution was added to each well. After incubation at room temperature for 30 min, the reaction was terminated by adding 50 µL stopping solution. The UV absorbance was recorded using Labsystems 96-well plate reader in dual-wavelength mode (450–650 nm), and the inhibitions were calculated. Finally, the 96-well plate was eluted with 300 mL methanol–acetic acid (7:1, v/v) for 2.0 h and 300 mL methanol for 1.0 h respectively for the next BELISA procedure.

Sample preparation

To check the accuracy of this developed BELISA method, the potato sample for spiking was determined to be free of acrylamide by GC. Briefly, 2.0 g of blank potato was separately weighed into a 50 mL conical flask, and then spiked with 0.5 mL standard acrylamide solution (50 mg L−1, 25 mg L−1 and 10 mg L−1). After incubated for 3.0 h, the spiking samples were ultrasonicly extracted with 3 × 10 mL of PBS for 30 min. The resulting extractions were collected and constant their volume to 50 mL by PBS. After filtered with 0.45 µm membrane, the filtrates were analysed by BELISA.

The French fries and cracker samples were prepared following the above steps except the addition of the acrylamide standard solutions, and analysed by the BELISA method.

Gas chromatography validation

For validating the accuracy of the developed BELISA method, the French fries and cracker samples were also determinate by GC. Briefly, 2.0 g of French fries or cracker samples were separately weighed into a 50 mL conical flask, and ultrasonicated with 3 × 10 mL DDW for 20 min. The resulting extractions were collected and centrifuged at 12 880 × g for 10 min, and the supernatants were merged and constant volume to 50 mL. Five millilitres of extracts and 1.0 mL sulfuric acid (10%, v/v) were sequentially added into a tube, and then placed in refrigerating cabinet (4°C) for pre-cooling 15 min. An aliquot of derivatisation reactants, including 1.0 mL of 0.1 mol L−1 potassium bromate and 1.50 g of potassium bromide powder, were added to the pre-cooled solution. The tubers were shaken by a vortex blender, and the reaction mixture was allowed to stand for 150 min at 4°C. The derivatisation reaction was terminated by adding 1.0 mL of 0.1 mol L−1 sodium thiosulfate. The solution was extracted three times with 10 mL ethyl acetate. The combined extracts were dried by sodium sulfate, filtered through a 0.45 µm membrane, and then injected into the gas chromatograph for analysis.

Preparation of the hydrophilic imprinted membrane

In an aqueous environment, the specific recognition between the MIPs and the template molecule can be weakened. Therefore, the sensitivity of the biomimetic immunoassay method is lower than traditional ELISA, which uses a biological antibody.

In order to improve the sensitivity of BELISA, preparation of a hydrophilic imprinted membrane compatible with an aqueous environment was investigated in this study. The results showed that when water was used as the solution, little molecularly imprinted membrane was grafted onto the 96-well plate wells. With the addition level of acetonitrile increasing, the thickness of the imprinted membrane was increased. Better results were observed when a mixture of 8 mL water and 12 mL acetonitrile was used as the reaction solution (Fig. 2).

image

Figure 2. The BELISA standard curves using a non-imprinted membrane prepared in water and acetonitrile, an imprinted membrane prepared in water and acetonitrile, and an imprinted membrane prepared in methanol as antibody, respectively.

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Structure analysis

In order to investigate the structures, the imprinted and non-imprinted membranes were isolated from the bottom of the wells and visualised by using scanning electron microscopy (SEM). They are depicted in Fig. 3. The SEM images show that they both possessed uniform and smooth surfaces, indicating that the imprinted membrane had been successfully prepared.

image

Figure 3. SEM images of novel imprinted membrane (a) and non-imprinted membrane (b) (×5000).

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In this study, the imprinted polymer was synthesised in a membrane format and the binding sites were situated on the surface, thus the enzyme conjugate can be accessed to them more easily. Furthermore, this hydrophilic imprinted membrane was directly synthesised on the 96-well plate wells, and the operation procedure of BELISA method was simplified.

Hapten selection

Enzyme (HRP) is a macromolecule, and the structure of enzyme-labelled antigen is quite different from the acrylamide, making the competitive reaction poor. Previous studies indicated that the labelled conjugate played an essential role in the specific recognition of MIP and affected the sensitivity of BELISA.[36] Acrylamide contains two reactive groups with a double bond and an amido link, which is ideal for chemical modification to synthesise the hapten for conjugating to enzyme. In order to obtain the HRP-labelled conjugate having the best performance in a MIP-based immunoassay, different linking handles (alkyl chain with different atoms) and position were investigated in this study.

The sensitivities of BELISA using enzyme conjugate A (3C), B (4C), C (8C) and D (double bond) were studied. The standard curves for four enzyme conjugates are shown in Fig. 4a and b. Results showed that enzyme conjugate C had a broader inhibition to acrylamide, from 3% to 55%, a higher sensitivity (IC50) and a lower limit of detection (LOD, IC15) than the other enzyme conjugates. So enzyme conjugate C was chosen for the next experiments. Results also indicated the link handle and position had important effects on the competitive reaction. If it is appropriate to extend the connection chain length, the sensitivity of BELISA will be improved. This might because the structural effect of enzyme on the selectivity of imprinted membrane toward enzyme-labelled antigen would be weaker when the connection chain (carbon chain) between the enzyme and hapten was appropriately extended.

image

Figure 4. The BELISA standard curves of acrylamide using enzyme conjugates of A, B, C and D, respectively, in which the vertical axes are inhibition (a) and absorbance (b).

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Optimisation of the BELISA conditions

In order to achieve optimal sensitivity and precision for the ELISA method, the parameters, including the enzyme conjugate concentration, the solvent composition and pH, were optimised in detail.

The enzyme conjugate concentration was determined by the titre of enzyme tracers to yield absorbance values ranging from 0.7 to 1.2. In this study, the enzyme conjugate was diluted to 1:3000 before use.

The solvent used for the preparation of standard solution and samples could affect on the sensitivity of the developed ELISA method. In order to investigate the influence on the assay performance, different working solutions, including 5% methanol aqueous solution, PBS solution, carbonate buffer solution (CBS) and water, were tested by comparing the inhibition and IC50 values obtained from corresponding standard curves. The results showed that PBS solution could provide a higher sensitivity for this method than the other solutions, and the IC50 value was 8.0 ± 0.4 mg L−1. Thus, PBS was chosen as the preparing solution for routine analysis of acrylamide.

Different pH values of PBS solution were also tested in the range of 2.91–11.07 to improve the sensitivity of the BELISA method (Fig. 5). The results showed that lower limit of detection and higher inhibition of the standard curve was obtained when the pH was around neutrality. This was because under conditions that are either too acidic or too alkaline the stability of the enzyme and acrylamide would be affected. Thus, the PBS solution in pH 7.02 was selected as the preparation solution in the following experiments.

image

Figure 5. The BELISA standard curves of acrylamide using PBS as preparation solution at different pH.

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Specificity of ELISA

Cross-reactivity studies were carried out by measuring the competitive curves and selectivity properties for other chemically related compounds under the optimal conditions. Cross-reactivity (CR) was calculated as the percentage between the IC50 value (calculated as the concentration giving 50% inhibition of colour development) for acrylamide and the IC50 value for the interfering compound with the following equation:

  • display math

The competitive assays of acrylamide between the related compounds are depicted in Table 1 and Fig. 2. It was shown that the biomimetic antibody (imprinted membrane) had a higher selectivity for acrylamide than the other related compounds, and the CR values of glycinamide hydrochloride, acrylic acid and propionamide were all less than 16%. We also observed that the standard curve using the imprinted membrane as the biomimetic antibody had a higher sensitivity and lower LOD than that of the non-imprinted membrane. A higher inhibition of 55.0% was obtained at 50 mg L−1, whereas only 22.8% inhibition was obtained for the non-imprinted membrane due to the non-specific adsorption, indicating that the imprinted membrane exhibited high selectivity for acrylamide.

Table 1. Structure, IC50 and cross-reactivity of the acrylamide and structurally related compounds of glycinamide hydrochloride, acrylic acid and propionamideThumbnail image of

Results in Fig. 2 also indicated that the BELISA method using the imprinted membrane prepared in water and acetonitrile as biomimetic antibody had better results than that of the imprinted membrane prepared in methanol, which indicated that the hydrophilic imprinted membrane exhibited improved recognition ability in PBS solution.

Parameters of the BELISA method

Under the optimised experimental conditions, the standard curve using the imprinted membrane as artificial antibody with varying concentrations of acrylamide from 16.0 µg L−1 to 50.0 mg L−1 in PBS solution was achieved (Fig. 4a). The LOD (IC15) of the developed BELISA method, calculated as the concentration of standard solution causing 15% inhibition of colour development, was 85.0 ± 4.2 µg L−1 and sensitivity (IC50) was 8.0 ± 0.4 mg L−1. Furthermore, this BELISA method had a broad detection range. These results demonstrated that this developed BELISA method is effective proof for the detection of a wide range of acrylamide levels in food samples.

Accuracy evaluation and sample analysis

In order to evaluate the accuracy of the presented BELISA method, the blank potato spiked with acrylamide at 100, 250 and 500 µg L−1 levels were determined. For each concentration, five measurements were performed, and the recoveries were 90.0 ± 7.2%, 104.0 ± 5.6% and 110.5 ± 5.0%, respectively. Moreover, the results also indicated that there was little matrix effect in the analysis of real sample using this developed BELISA method without any other sample treatment procedure except filtration. Thus, the pretreatment was simplified.

It is known that French fries and cracker are typical starch-based foods, and a large amount of acrylamide can be produced during the deep-heating process. The applicability of this developed BELISA method was validated by comparative analysis of the French fries and cracker samples with GC (Table 2). The results obtained by these two methods were correlated well and there are no significant differences (P > 0.05). Moreover, the acrylamide in French fries and cracker samples was quantitatively detected by the BELISA method with different levels of 0.440 ± 0.016 mg kg−1 and 0.424 ± 0.028 mg kg−1, respectively. However, WHO and the European Union have limited the content of acrylamide in drinking water to 0.5 µg L−1 and 0.1 µg L−1, respectively. Such a high level of acrylamide in the French fries and cracker is, potentially, a serious harm to human health. Therefore, further research should be devoted to the control of acrylamide formation in the heating food products.

Table 2. Comparison of concentrations of acrylamide in French fries and cracker samples analysed by BELISA and GC methods
SampleAnalysis results (mg kg−1, ± SD)PSignificant difference
BELISAGC
French fries0.440 ± 0.0160.465 ± 0.0290.127None
Crackers0.424 ± 0.0280.438 ± 0.0310.423None

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In this study, the developed BELISA method was sensitive and simple due to the high selectivity of the biomimetic antibody (imprinted membrane). The LOD of the BELIA method was almost the same as the reported result (65.7 µg kg−1) of Preston and Fodey[15] which was obtained by the direct competitive ELISA method using a biological antibody. This might result from the imprinting effect, the difference of the molecular interactions and their structures. During the preparation of the novel imprinted film, the COOH of MAA was reacted with the NH2 of acrylamide, and the imprinted cavities and specific binding sites for the hydroxyl group in a predetermined orientation were generated.

Furthermore, without antibody coating and BSA/PBS blocking procedure, the operation time was reduced to that of the traditional ELISA. The imprinted membrane can be re-used 20 times without loss of sensitivity, and the cost per analysis of this developed method was drastically reduced. Therefore, the developed method has many merits for rapid detection of acrylamide.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In this paper, an improved BELISA method based on a hydrophilic imprinted membrane was developed for the determination of acrylamide in French fries and cracker samples. With the properties of high sensitivity, pre-treatment simplicity and low cost, this proposed method could be a promising screening methodology in food sample analysis. Moreover, the results shown in this paper will help lead to further research on MIP-based immunoassays.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The authors are grateful for financial support from the National Natural Science Foundation of China (project No. 31071543) and the Science & Technology Project of Taian, China (20123064).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. DISCUSSION
  6. CONCLUSIONS
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  • 1
    Ahn JS, Castle L, Clarke DB, Lloyd AS, Philo MR and Speck DR, Verification of the findings of acrylamide in heated foods. Food Addit Contam 19:11161124 (2002).
  • 2
    Riediker S and Stadler RH, Analysis of acrylamide in food by isotopedilution liquid chromatography coupled with electrospray ionisation tandem mass spectrometry. J Chromatogr A 1020:121130 (2003).
  • 3
    Rosén J and Hellenaes KE, Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry. Analyst 127:880882 (2002).
  • 4
    Senyuva HZ and Gkmen V, Survey of acrylamide in Turkish foods by an in-house validated LC-MS method. Food Addit Contam 22:204209 (2005).
  • 5
    Wenzl T, Beatriz M, Calle DL and Anklam E, Analytical methods for the determination of acrylamide in food products: A review. Food Addit Contam 20:885902 (2003).
  • 6
    Swedish National Food Administration, Information about acrylamide in food. Swedish National Food Administration, Stockholm, 2002.
  • 7
    Costa LG, Deng H, Gregotti C, Manzo L, Faustman EM, Bergmark E, et al., Comparative studies on the neuro and reproductive toxicity of acrylamide and its epoxide metabolite glycidamide in the rat. Neuro Toxic 13:219224 (1992).
  • 8
    Dearfield KL, Douglas GR, Ehling UH, Moore MM, Sega GA and Brusick DJ, Acrylamide: A review of its genotoxicity and an assessment of heritable genetic risk. Mutat Res 330:7199 (1995).
  • 9
    International Agency for Research on Cancer (IARC), Some Industrial Chemicals, IRAC Monographs on the Evaluation of Carcinogenic Risk to Humans no. 60. IARC, Lyon, pp. 389441 (1994).
  • 10
    Friedman M, Chemistry, biochemistry and safety of acrylamide. A review J Agric Food Chem 51:45044526 (2003).
  • 11
    Zhang Y, Dong Y, Ren YP and Zhang Y, Rapid determination of acrylamide contaminant in conventional fried foods by gas chromatography with electron capture detector. J Chromatogr A 1116:209216 (2006)
  • 12
    Alain P, Adrienne P and Jean MO, Trace level determination of acrylamide in cereal-based foods by gas chromatography–mass spectrometry. J Chromatogr A 1035:123130 (2004).
  • 13
    Cheong TK, Eun SH and Hyong JL, An improved LC-MS/MS method for the quantitation of acrylamide in processed foods. Food Chem 101:401409 (2007).
  • 14
    Fu YJ, Li Q, Chen JY, Wang L, Li R, Zhou GP, et al., Rapid detection of acrylamide residue in heated food by enzyme linked immunosorbent assay. Chin Brew 230:7779 (2011).
  • 15
    Preston A and Fodey T, Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis. Anal Chim Acta 608:178185 (2008).
  • 16
    Zhang HX, Gao MQ, Zhang XR and Liu H, Synthesis of acrylamide artificial antigen and preparation of anti-acrylamide polyclonal antibody. Chin Agric Sci Bull 25:8385 (2009).
  • 17
    Esteban M, Molecular imprinting technology: a simple way of synthesizing biomimetic polymeric receptors. Anal Chim Acta 378:1875 (2004).
  • 18
    Lv YK and Yan XP, Preparation and application of molecularly imprinted sol–gel materials. Chin J Anal Chem 33:254260 (2005).
  • 19
    Matthew PD, Vern DB and David P, Approaches to the rational design of molecularly imprinted polymers. Anal Chim Acta 504:714 (2004).
  • 20
    Vivek BK and Hunag J, Molecular imprinting: a dynamic technique for diverse applications in analytical chemistry. Anal Bioanal Chem 380:587605 (2004).
  • 21
    Hu ML, Jiang M, Wang P, Mei SR, Lin YF, Hu XZ, et al., Selective solid-phase extraction of tebuconazole in biological and environmental samples using molecularly imprinted polymers. Anal Bioanal Chem 387:10071016 (2007).
  • 22
    Bravo JC, Garcinuo RM, Fernández P and Durand JS, A new molecularly imprinted polymer for the on-column solid-phase extraction of diethylstilbestrol from aqueous samples. Anal Bioanal Chem 388:1039 (2007).
  • 23
    Li CY, Wang CF, Wang CH and Hu SH, Construction of a novel molecularly imprinted sensor for the determination of O,O-dimethyl-(2,4-dichlorophenoxyacetoxyl) (3′-nitrophenyl) methinephosphonate. Anal Chim Acta 545:122128 (2005).
  • 24
    Hillberg AL, Brain KR and Allender CJ, Molecular imprinted polymer sensors: Implications for therapeutics. Adv Drug Deliver Rev 57:18751889 (2005).
  • 25
    Kitade T, Kitamura K, Konishi T, Takegami S, Okuno T, Ishikawa M, et al., Potentiometric immunosenor using artificial antibody based on molecularly imprinted polymers. Anal Chem 76:68026807 (2004).
  • 26
    Hall AJ, Emgenbroich M and Sellergren B, Imprinted polymers. Top Curr Chem 249:317349 (2005).
  • 27
    Li ZL, Wang S, Lee NA, Allan RD and Kennedy IR, Development of a solid-phase extraction-enzyme-linked immunosorbent assay method for the determination of estrone in water. Anal Chim Acta 503:171177 (2004).
  • 28
    Fang GZ, Lu JP, Pan MF, Li W, Ren L and Wang S, Substitution of antibody with molecularly imprinted film in enzyme-linked immunosorbent assay for determination of trace ractopamine in urine and pork samples. Food Anal Method 4:590597 (2011).
  • 29
    Meng L, Qiao XG, Xu ZX, Xin JH and Wang L, Development of a direct competitive biomimetic enzyme-linked immunosorbent assay based on a hydrophilic molecularly imprinted membrane for the determination of trichlorfon residues in vegetables. Food Anal Method 5:12291236 (2012).
  • 30
    Palomo C, Aizpurua JM, Legido M and Galarza R, N-Methylidene [bis(trimethylsilyl)methyl]amine: the first isolable and stable monomeric methanimine allowing thermal [2 + 2] cycloadditions with ketenes. Anal Commun 34:233234 (2011).
  • 31
    Haupt K, Dzgoev A and Mosbach K, Herbicide assay using an imprinted polymer based on system analogous to competitive fluoroimmunoassay. Anal Chem 70:628631 (1998).
  • 32
    Haupt K, Mayes AG and Mosbach K, Herbicide assay using an imprinted polymer based on system analogous to competitive fluoroimmunoassay. Anal Chem 70:39363939 (1998).
  • 33
    Haupt K and Mosbach K, Plastic antibodies: developments and applications. Trends Biotechnol l16:468475 (1998).
  • 34
    Ellwanger A, Berggren C, Bayoudh S, Crecenzi C, Karlsson L and Owens PK, Evaluation of methods aimed at complete removal of template from molecularly imprinted polymers. Analyst 126:784792 (2001).
  • 35
    Xing SJ and Chen FS, Synthesis of oxytetracycline artificial antigen. Food Sci 26:242245 (2005).
  • 36
    Xu ZX, Gao HJ, Zhang LM, Chen XQ and Qiao XG, The biomimetic immunoassay based on molecularly imprinted polymer: A comprehensive review of recent progress and future prospects. Food Sci 77:R69R75 (2011).