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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 1  |  Issue : 2  |  Page : 109-113

Detection of TNT by Surface Plasmon Resonance Based on Molecularly Imprinted Polymers


1 Key Laboratory of Evidence Science, University of Political Science and Law, Ministry of Education; Institute of Forensic Science, Ministry of Public Security, Beijing, China
2 Key Laboratory of Evidence Science, University of Political Science and Law, Ministry of Education, Beijing, China
3 Key Laboratory of Evidence Science, University of Political Science and Law, Ministry of Education; Collaborative Innovation Center of Judicial Civilization, China

Date of Web Publication27-Nov-2015

Correspondence Address:
Hongxia Hao
Key Laboratory of Evidence Science, China University of Political Science and Law, Ministry of Education, Beijing - 100088
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2349-5014.162780

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  Abstract 

2,4,6-trinitrotoluene (TNT) is a commonly used explosive. It is not only a threat to public safety but also causes environmental pollution, affecting human health. However, at this stage of TNT detection, technology cannot meet the demands of the current situation. To acquire a new method devoted to the study of fast and quantitative detection of TNT. It combines the molecular imprinting technique (MIT) with surface plasmon resonance (SPR) technology for high sensitivity. In this study, a molecularly imprinted polymer (MIP) film for the detection of TNT was synthesized by heat in acetonitrile at 60°C, using the TNT imprinting molecule and azobisisobutyronitrile (AIBN) as initiators. In the present work, there are many factors that can influence the elution efficiency, such as raw material ratios,fore-reaction time, reaction time, etc. The polymers have the highest elution efficiency when raw material mole ratios [n(TNT):n methacrylic acid (MAA):n ethylene glycol dimethylacrylate (EGDMA)] were 1:4:8; the MIP sensor could detect a TNT concentration as low as 1×10-10 M. Compared to the blank polymer with the same chemical composition, the imprinted polymer had higher binding efficiency and higher selectivity.

Keywords: Molecularly imprinted polymer, 2,4,6-trinitrotoluene, surface plasmon resonance


How to cite this article:
Zhou H, Zeng L, Chen X, Hao H. Detection of TNT by Surface Plasmon Resonance Based on Molecularly Imprinted Polymers. J Forensic Sci Med 2015;1:109-13

How to cite this URL:
Zhou H, Zeng L, Chen X, Hao H. Detection of TNT by Surface Plasmon Resonance Based on Molecularly Imprinted Polymers. J Forensic Sci Med [serial online] 2015 [cited 2019 Sep 15];1:109-13. Available from: http://www.jfsmonline.com/text.asp?2015/1/2/109/162780


  Introduction Top


2, 4, 6-trinitrotoluene (TNT) is commonly found in explosive cases. It is not only a threat to public security but also causes environmental pollution and endangers health. The traditional TNT detection methods are gas chromatography,[1] high-performance liquid chromatography,[2] electrochemical analysis,[3],[4],[5] and chemically modified electrode method.[6],[7] However, these methods have drawbacks such as poor specificity, long testing cycles, and complex pretreatment protocols, which cannot meet the needs required by actual sample handling; therefore, developing a method for on-site rapid detection of TNT will be necessary.

Molecular imprinting technique (MIT) is a new multidisciplinary technology originating from Fischer's [8] proposed "keys theory" in 1894. MIT is also known as the molecularly imprinted polymers (MIPs) experimental technique,[1],[9] which is applied to synthesizing polymers, meeting spatial structure, and binding sites for an exact match with the template molecule with high selectivity. The basic principle of MIT is making the template and the functional monomer participate in the polymerization reaction of a crosslinking agent. After completing the reaction, the template molecules are removed by physical and chemical methods and consequently, a number of binding sites are left on the position of the original template molecule. These binding sites can identify a particular type of molecule because they possess the same species specificity of recognition.

Surface plasmon resonance (SPR) is determined by measuring the change in refractive index near the metal surface to study the nature of the substance. The theory is based on attenuated total reflection (ATR).[3] Both Otto [2] (in the late 1960s) and Kretschmann [4] (in 1971) published articles expressing the observed phenomenon of SPR by the attenuated total reflection method; the contribution of these two scholars is considered a landmark breakthrough in the field of SPR research. Currently, the scientific community has successfully developed a variety of SPR sensors for biological and chemical detection for application in many diverse areas. Practice has demonstrated that SPR sensors have many advantages when compared to conventional testing methods, such as no requirement for sample labeling, real-time motion detection, and high sensitivity. Therefore, SPR sensors have broad application prospects in medical diagnostics, environmental monitoring, biotechnology, pharmaceutical development, food safety inspection, and other fields.[5],[6],[7],[10],[11],[12],[13],[14],[15],[16],[17]

In this paper, TNT-imprinted polymer films were characterized by SPR detection in order to meet the needs of high sensitivity, high stability, and high selectivity present in TNT detection. This method is also markedly different from the method used by Han et al.[18] In the synthesis process of nanometer gold film, the detection limit is much lower compared with the research of Han et al. Furthermore, based on the research in detecting TNT, we expect to develop sensor chips to meet the needs of on-site rapid explosives detection in the actual cases.


  Materials and Methods Top


Reagents and equipment

Reagents

TNT, standard materials (provided by the Institute of Forensic Science, China), Methacrylic acid(MAA), AR (Tianjin Bodi Chemical Co., Ltd) (prior to use by vacuum distillation to remove inhibitor).

Ethylene glycol dimethylacrylate (EGDMA), AR, US (prior to use by vacuum distillation to remove inhibitor).

Azobisisobutyronitrile (2, 2'-Azobisisobutyronitrile) (AIBN) chemically pure, Beijing Chemical Reagent Co., Ltd.

Trimethylchlorosilane, AR, Sinopharm Chemical Reagent Co., Ltd.

Hydrogen peroxide (30%), AR, Beijing North Fine Chemical Co., Ltd.

Concentrated sulfuric acid (98%), AR, Beijing North Fine Chemical Co., Ltd, acetonitrile, AR, Beijing North Fine Chemical Co., Ltd.

Alcohol, AR, Sinopharm Chemical Reagent Co., Ltd.

Dimethyl sulfoxide (DMSO), AR, Beijing North Fine Chemical Co., Ltd.

Equipment

Electronic balance (AR1140, Ohaus U.S. International Trade Co., Ltd.).

Collector constant temperature heating magnetic stirrer (DF-101S, Zhengzhou Great Wall Industry and Trade Co., Ltd.).

Ultrasonic cleaning machine (KS-180EII, Ningbo Branch of Health Ultrasound Equipment Co., Ltd.).

SPR instrument (self-assembly).

Varian 50 Conc UV-visible spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.).

Synthesis of TNT MIP

Steamed plating gold film in vacuum

Lanthanum (La) glass should be washed before coating; the following methods for coating were adopted:First, La glass was placed in a beaker and washed with detergent for 20 min in an ultrasonicator followed by deionized water for 20 min and lastly, rinsed in ethanol before drying the La glass dry by forced nitrogen. If the La glass was difficult to clean, a mixture of sulfuric acid and hydrogen peroxide was used to clean the glass first followed by the cleaning protocol initially described.

Pretreatment of the slides

The slides were sonicated using detergent for 20 min and then placed in deionized water to soak for 20 min. Next, the slides were placed in concentrated H2 SO4:H2O2 [3:1, volume/volume (V/V)] solution for about 1 h. After removal, they were rinsed with deionized water and ethanol, and finally dried in the oven or under forced nitrogen. They were then placed in 50 mM trimethylchlorosilane in anhydrous ethanol for 24 h to silanize the glass surface to reduce and prevent the polymer from adhering to the surface. The configured silane in ethanol was sealed and protected from contamination for further reuse.

Purification of the initiator AIBN

AIBN initiator was purified by recrystallization; the protocol is described below:

  • To dissolve, ethanol was added to a 200 mL Erlenmeyer flask, then heated to near boiling using a water bath (about 80°C) followed by the immediate addition of about 20 g of AIBN after which the AIBN dissolved. If the dissolution process is slow, the Erlenmeyer flask can be gently shaken to assist in dissolution
  • Filtration (note: Hot filtration should be adopted using a Büchner funnel): When the flask was cooled to room temperature, white crystals began to appear
  • To dry, the white crystals were placed in Petri dishes to air-dry for 24 h, followed by vacuum drying
  • Storage: After purification, AIBN was stored and set aside in a cool and dry environment away from natural light. AIBN should be stored properly in order to prevent contamination.


Purification of MAA and EGDMA

Commercial MAA and EGDMA products contain inhibitors, which can lead to spontaneous reaction in the polymerization. During transportation and storage, MAA and EGDMA must be purified by the method of vacuum distillation prior to use. The refined products should be stored at 4°C away from light.

Experimental conditions

After multiple experiments, MAA was selected as the monomer, along with acetonitrile: DMSO = 9:1 (volume ratio) as the solvent mixture, EGDMA as the crosslinking agent, and lastly, AIBN as the initiator. The following conditions were used: TNT: MAA: EGDMA = 1:4:8 (substance ratio), acetonitrile: DMSO = 9:1 (volume ratio), constant water bath temperature of 65°C, and a reaction time of 24 h. The aforementioned conditions produced a molecularly imprinted polymeric membrane for TNT with good performance.


  Results and Discussion Top


Characterization of TNT molecularly imprinted polymer (MIP) membrane by SPR

TNT molecularly imprinted polymeric membrane required instrumental evaluation of its performance. In the present work, a self-assembled SPR instrument was set up and used for detection. Further attention was given to the study on elution and adsorption of the TNT MIP, obtaining a linear equation, R2 = 0.9730. With this equation, the unknown sample was quantitatively analyzed.

SPR characterization of MIP membrane in various media

A TNT: MAA: EGDMA molar ratio of 1:4:8 was used to synthetize the MIP and 3 mL acetonitrile was used as the solvent. Then the MIP membrane synthesis was carried out in acetonitrile or air for SPR detection; the SPR reflectivity results are shown in [Figure 1].
Figure 1: The SPR spectra comparison of MIP films synthesized in acetonitrile (solid) and air (hollow)

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[Figure 1] shows that the resonance angle of the MIP membrane synthesis in air is 77.43°, while the SPR resonance angle spectrum of the membrane in acetonitrile is 75.19°. Although the two membranes are of the same system, the SPR angle varies between the solid and flowing liquid phases. Comparatively speaking, the resonance angle is more stable in the liquid medium (acetonitrile). Furthermore, the resonance angle is smaller and thus, better for the experimental analysis.

Dynamics study on elution of nonTNT MIP membrane

The dynamics research on elution of nonTNT molecularly imprinted membrane (blank)

The concentration ratio of monomer and crosslinking agent, MAA: EGDMA, used in this experiment was 1:2 and the volume ratio of acetonitrile and DMSO was 9:1 for preparation of the gold film that does not contain the TNT imprinted polymer membrane on the glass slide (blank). The study of elution dynamics of the nonTNT MIP membrane was then performed by the self-assembled SPR instrument; the results are shown in [Figure 2].
Figure 2: Elution kinetics curve of nonTNT MIP membrane

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As shown in [Figure 2], the scanning curve will tend to be smooth at the 10th min. After injecting 90% of the eluent (volume ratio of solvent to acetic acid is 9:1), the reflected light intensity increases since the eluent increases the refractive index and eventually, the reflected light intensity is stabilized. Following injection of the solvent, there is no substantial difference in light intensity compared to that before elution when the dynamic curve was stable.

Dynamics study on elution of TNT MIP membrane

The concentration of TNT for the template was chosen to be 0.05 mmol and using a mixture of acetonitrile and DMSO as the solvent for the synthesis of the TNT MIP membrane on the gold surface of the chip, the molar ratio of TNT: MAA: EGDMA was 1:4:8. The elution dynamics were studied using the self-assembled SPR instrument; the results are shown in [Figure 3] and [Figure 4].
Figure 3: Elution dynamics curves of TNT MIP film

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Figure 4: The SPR spectra comparison of MIP films before and after elution

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[Figure 3] shows the dynamic curve of solvent were stable after the 8th min. Upon adding the configured eluent (volume ratio of solvent to acid is 9:1), the reflected light intensity was reduced by about 10%, due to the elution of the TNT from the membrane, forming the template pores. Overall, the refractive index from blank elution experiments was greater than that in acetonitrile. The time-intensity curve shows that the curve is stabilized at the 30th min and after adding the solvent, the kinetic curve declined by about 2% and gradually became smooth. After elution, the scan spectra show that the resonance angle decreased by approximately 1.75° when compared with the resonance angle before elution. This demonstrates that the acetate eluent disrupts the chemical forces between TNT and MAA, breaking the hydrogen bonds. Thus, TNT molecule can be eluted from the polymer molecules, forming a hole in the three-dimensional structure. Thus, it can be said that the molecularly imprinted membrane will produce changes in the resonance angle after elution.

[Figure 4] shows that the resonance angle decreased and the resonance depth increases after elution. This was due to the removal and elution of the TNT in the MIPs, resulting the formation of three-dimensional pores that were replaced with solvent. The higher the refractive index of the solvent, greater the resonance depth and smaller the resonance angle. It is expected that in the next step of the elution process, the range between the resonance angles of the two extreme points should become larger. After elution, the formation of the three-dimensional holes in the MIP membrane causes a shift to the left of the resonance corner. Thus, we can infer that the resonance curve gradually shifted to the right with continuous absorption of TNT molecules.

SPR spectra of TNT absorption by MIP membrane

A series of solutions varying in concentration gradients of TNT in acetonitrile were prepared (10-16 M, 10-15 M... 10-5 M) to detect and monitor TNT adsorption using the TNT MIP membrane by SPR. The reflectivity was recorded on the vertical axis against the negative logarithm of the TNT concentration. The adsorption by the TNT MIP membrane is recorded followed by elution and cleaning, shown in [Figure 5],[Figure 6],[Figure 7],[Figure 8].
Figure 5: The molar concentration ratio of TNT:MAA:EGDMA is 1:4:8, volume of 3 mL of acetonitrile, TNT molecularly imprinted membrane synthesis SPR angle TNT 10−5 M ~ 10−16 M absorption spectra

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Figure 6: Details of spectral minima from Figure 5

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Figure 7: Linear regression of SPR resonance angle against negative logarithm of concentration

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Figure 8: Linear regression of the second resonance absorption SPR angle against the negative logarithm of TNT concentration

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As seen from [Figure 5] and [Figure 6], as the TNT concentration increased, the SPR resonance angle increased. For the entire concentration range (lowest to highest: 10-15 M... 10-5 M), the resonance angles were transformed from77.07° to 77.33°, 77.41°, 77.61°, 77.69°, 77.83°, 77.87°, 78.09°, and 78.37°, respectively. It is evident that the SPR sensor is capable of TNT detection.

[Figure 7] shows that within a certain range of concentrations, the TNT MIP membrane demonstrates a change in the resonance angle, with the concentration of TNT into a linear relationship with a correlation coefficient of 0.9730. [Figure 7] shows that within a certain range of concentrations, the TNT MIP membrane demonstrates a linear relationship between the resonance angle and the TNT concentration, with a correlation coefficient of 0.9730. Similarly, [Figure 8] shows the TNT MIP membrane secondary detection of TNT still retains this good linear relationship and behavior, with a correlation coefficient of 0.9599. Overall, it was shown that the employed method of TNT MIP membranes produced stable, highly reproducible surfaces that are capable of quantitative detection to trace TNT.


  Conclusion Top


The present article combined molecular imprinting technology with SPR to detect TNT. TNT template molecules were used to prepare MIP membranes for specific recognition and were later characterized using SPR technology. After elution of TNT template molecules from the MIP polymer film/membrane, three-dimensional holes were formed. The membrane was shown to have strong adsorption capacity for varying concentrations of TNT. The results showed that after elution, for a certain range of concentrations of TNT (10-15 M to 10-7 M) and absorption resonance angle showed a good linear relationship. The resulting linear regression equation, y = -0.1437x + 79.277 (R2 = 0.9730), could be used to infer the TNT content of a sample. The synthesis of TNT MIP membrane was shown to be stable and suitable for secondary use.

Acknowledgement

This work is one part of research on drug and development of explosive detection technique, and was supported by the Opening Project of Key Laboratory of Evidence Science (China University of Political Science and Law), Ministry of Education (2012KFKT07), the Program for Young Innovative Research Team in China University of Political Science and Law (1000-10814344), Academician Foundation of the Ministry of Public Security of the People's Republic of China (No. 2011-23210044, 2011-23211119, 23212052). The corresponding author is from the China Collaborative Innovation Center of Judicial Civilization.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]


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