Chemistry-An Asian Journal. 2016 Oct;11;3097-3101. dio: 10.1002/asia.201601079

Identification of Endocrine Disrupting Chemicals using a Virus-Based Colorimetric Sensor

 

Jong-Sik Moona, Yujin Leea,b, Dong-Myeong Shinc, Chuntae Kima,b, Won-Geun Kima,b, Minji Parkd, Jiye Hana,b, Hyerin Songe, Kyujung Kime, Jin-Woo Oha,b,c,f*

aBK21 PLUS Nano convergence Technology Division, Pusan National University, Busan 46241, Republic of Korea

bDepartment of Nano Fusion Technology, Pusan National University, Busan 46241, Republic of Korea

cResearch center for Energy Convergence Technology, Pusan National University, Busan 46241, Republic of Korea

dDepartment of Applied Nanoscience, Pusan National University, Busan 46241, Republic of Korea

eDepartment of Cogno-Mechatronics Engineering, Pusan National University, Busan 46241, Republic of Korea

fDepartment of Nanoenergy Engineering, Pusan National University, Busan 46241, Republic of Korea

Correspondence should be addressed to Jin-Woo Oh. E-mail: ojw@pusan.ac.kr

 

Abstract 

A simple and portable colorimetric sensor based on M13 bacteriophage (phage) was devised to identify class of endocrine disrupting chemicals, including benzene-, phthalic- and chlorobenzene-derivatives. Arrays of structurally, genetically modified, M13 bacteriophage were fabricated as to produce a biomimetic colorimetric sensor, and color changes of the phage arrays in response to several benzene derivatives were characterized. The sensor was also used to classify phthalic- and chlorobenzene derivatives representative of endocrine disrupting chemicals. The characteristic color patterns obtained on exposure to various benzene-derivatives enabled with similar chemical structures in vapor phase to be classified. Our sensing approach based on the use of a genetically surface modified M13 bacteriophage offers a promising platform for portable, simple environmental monitors, which could be extended for use in numerous application areas, including food monitoring, security monitoring, explosive risk assessment, and point of care testing.

 

Supplement

For decades, many kinds of endocrine disrupting chemicals (EDCs) such as phthalate and polychlorinated compounds (PCB) were commonly used to improve properties of polymer products. Recently, most of the endocrine disrupting additives in polymer product fabrication were regulated, however, respectable amount of EDCs have still been found in numerous merchandises including adhesives, inks, electrics, and paints. Due to the severe adverse effect on endocrine system by EDCs in central nervous system of human and wildlife, detecting and monitoring EDCs collect great attentions. Although many techniques have been developed including fluorescence, enzyme linked immune sorbent assays (ELISA), piezoelectric biosensing, chromatographic sensing, and electrochemical sensing; fast, simple and reliable sensing technique is strongly required.

 

M-13 bacteriophage have nano-rod shape with ~880 nm of length and ~6.6 nm of length, and composed of single strain DNA covered by 2,700 copies of helically arranged pVIII major coat protein on viral body. Since foremost major and minor coat protein of M-13 bacteriophage are structurally well known, genetic engineering of coat protein to reveal desired properties can be easily done. A genetically engineered M-13 bacteriophage-based nano-structure was fabricated by simple self-assembly technique and was used into colorimetric sensor. The M-13 bacteriophage-based colorimetric sensor with superior selectivity and sensitivity can be used in various chemical detection and discrimination such as EDCs, antibiotics, and distinctive VOCs from some cancer cells.

 

WHW type of M-13 bacteriophages was prepared by phage display technique and genetic engineering [1]. M-13 bacteriophage’s surface was modified to express a Tryptophan (W) – Histidine (H) – Tryptophan (W) – Glutamine (Q) sequence of amino acids in the pVIII major coat protein, replacing Ala-Glu-Gly-Asp-Asp residues in wild-type bacteriophage to Ala-AspAsp-Trp(W)-His(H)-Trp(W)-Gln(Q)-GluGly-Asp residues. Genetically modified protein sequence leads to increase in a selectivity of the benzene derivatives due to π-stacking interactions, which is abundant in target EDCs in this research, phthalate derivatives and PCBs.

 

Three arrays of structurally colored M-13 Bacteriophage nano-structure on Au coated Si wafer as colorimetric sensor was fabricated using simple pulling technique by using different pulling speed [1, 2]. M-13 bacteriophage arrays by preparing aqueous phage solution composed of smectic bundle structure of M-13 bacteriophage nanofilaments with different diameters and interspacings at each array. Through the dynamical change in the interspace of M-13 bacteriophage bundle structures, structural color change occurs instantly by exposure to target chemicals. All color change measurements were performed using a home-built M-13 bacteriophage-based colorimetric sensor detection system. Color changes of sensor with respect to real-time RGB signal upon exposure to target chemicals were detected with CCD camera which is fitted on a sealed chamber at desired temperatures. All data collection were controlled by MATLAB program [2-5].

 

Due to the self-assembly characteristics and genetic modification of protein structure on the surface of bacteriophage, the M-13 bacteriophage-based color sensor exhibits remarkable sensitivity and selectivity to detect minute amount of target chemicals. To validate the feasibility of M-13 bacteriophage-based colorimetric sensor for EDCs detection, phthalic anhydride and chlorobenzene were measured. Both chemicals were selected because they are basic structure of the following target EDCs. For the main measurement, M-13 bacteriophage-based colorimetric sensor was tested with various target EDCs such as phthalates (diethyl phthalate, dibutyl phthalate, benzyl-butyl-phthalate and bis-(2-ethylhexyl)-phthalate) and PCBs (PCB18, PCB101, PCB138, PCB180 and PCB209). Target materials were heated to produce VOCs and color change of colorimetric sensor were detected using CCD camera. All discrimination data were analyzed using principle component analysis (PCA) technique, and the first two discriminant factors account for 64.02%. Through linear discrimination analysis (LDA), LDA error of EDCs was 3.7%. Sensitivity of M-13 bacteriophage-based colorimetric sensors were analyzed using color difference of sensor before and after detection. M-13 bacteriophage-based colorimetric sensor showed the significant performance upon the exposure to target material concentration of 10 ppm. The above mentioned results suggest M-13 bacteriophage-based nano-structure as an important platform in colorimetric sensors and thereby leading to potential application in various fields, such as, portable environmental monitoring and point of care diagnostic devices.

 

 

Figure 1. Sensitive and selective sensing of EDCs using the M-13 bacteriophage-based colorimetric sensor. a) Characteristic color patterns of the colorimetric sensor after exposure to different concentrations of phthalic anhydride from 50 to 300 ppm. b) Color pattern of the M13 bacteriophage-based colorimetric sensor after exposure to different chlorobenzene concentrations (from 10 to 200 ppm). Color changes of M-13 bacteriophage-based colorimetric sensor triggered upon exposure to VOCs from target chemicals by changing interspace of M-13 bacteriophage bundle structure. c) Selective sensing ability of M-13 bacteriophage-based colorimetric sensor by exposure to various EDCs such as PCBs and d) phthalates, as reported Chem. Asian J. 11: 3097-3101 (2016).

 

 

Figure 2. Discrimination of representative EDCs using M-13 bacteriophage-based colorimetric sensor, including phthalates and PCBs. a) PCA plot of the color changes of M-13 bacteriophage-based color sensor resulting from exposure to phthalates. The data in the PCA plot was taken from sensor color patterns after exposure to 28.5 ppm of phthalate derivative VOCs. b) PCA plot of the color changes resulting from exposure to PCBs and phthalates. The PCBs data in the PCA plot was taken from sensor color patterns after exposure to 5 mg of PCBs. Solid PCBs were evaporated by heating at 30 ºC for 10 minutes in sealed chamber. Linear discriminant analysis (LDA) plots clearly discriminate PCBs and phthalates, as reported Chem. Asian J. 11: 3097-3101 (2016).

 

References

[1] Woo-Jae Chung, Jin-Woo Oh, Kyungwon Kwak, Byung Yang Lee, Joel Meyer, Eddie Wang, Alexander Hexemer, and Seung-Wuk Lee. “Biomimetic self-templating supramolecular structures,” Nature, vol. 478, no. 7369, pp. 364-368, 2011
[2] Jin-Woo Oh, Woo-Jae Chung, Kwang Heo, Hyo-Eon Jin, Byung Yang Lee, Eddie Wang, Chris Zueger, Winnie Wong, Joel Meyer, Chuntae Kim, So-Young Lee, Won-Geun Kim, Marcin Zemla, Manfred Auer, Alexander Hexemer, and Seung-Wuk Lee “Biomimetic virus-based colourimetric sensors,” Nature Communications, vol. 5, no. 3043, doi: 10.1038/ncomms4043, 2013
[3] So-Young Lee, Jong-Sik Moon, Dong-Myeong Shin, Chuntae Kim, Won-Geun Kim, Kyujung Kim, So Young Yoo, and Jin-Woo Oh. “Bioinspired M-13 bacteriophage-based Photonic Nose for Differential Cell Recognition,” Chemical Science, vol. 8, pp. 921-927, 2017
[4] Jong-Sik Moon, Minji Park, Won-Geun Kim, Chuntae Kim, Jinyoung Hwang, Daun Seol, Chang-Seok Kim, Jong-Ryeul Sohn, Hoeil Chungd, and Jin-Woo Oh. “M-13 bacteriophage based structural color sensor for detecting antibiotics,” Sensors and Actuators B: Chemical, vol. 240, pp. 757-762, 2017
[5] Jong-Sik Moon, Yujin Lee, Dong-Myeong Shin, Chuntae Kim, Won-Geun Kim, Minji Park, Jiye Han, Hyerin Song, Kyujung Kim, and Jin-Woo Oh. “Identification of Endocrine Disrupting Chemicals using a Virus-Based Colorimetric Sensor,” Chemistry-An Asian Journal, vol. 11, pp. 3097-3101, 2016