Colorimetric Detection of Carcinogenic Aromatic Amine Using Layer

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Surfaces, Interfaces, and Applications

Colorimetric Detection of Carcinogenic Aromatic Amine Using Layer-by-Layer Graphene Oxide/ Cytochrome C Composite Zhi-bei Qu, Ling-fei Lu, Min Zhang, and Guoyue Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01176 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 13, 2018

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ACS Applied Materials & Interfaces

Colorimetric Detection of Carcinogenic Aromatic Amine Using Layer-by-Layer Graphene Oxide/ Cytochrome C Composite Zhi-bei Qua,b, Ling-fei Lua, Min Zhanga and Guoyue Shia* a. School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China. b. Department of Chemical Engineering, University of Michigan, Ann Arbor MI 48105, USA.

CORRESPONDING AUTHOR FOOTNOTE. * Prof. Guoyue Shi, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China; Tel./Fax: (+86) 021-54340043; Email: [email protected]

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ABSTRACT. Graphene and its derivatives were found to be efficient modulators of enzymes in various systems. However, the modulating mechanism was not well discussed for long time. Inspired by the artificial enzyme enhancing property of graphene oxide (GO) towards cytochrome c (cyt. c), we have successfully fabricated a protein/GO hybrid structure via a layerby-layer (LbL) strategy. The obtained LbL assemblies showed great enhancement in peroxidase activity of cyt. c, as well as excellent stability, resistance to extreme environment change, and also possibility for recycling by simple centrifugation without any obvious activity loss. The LbL cyt. c/GO hybrids were expanded to a colorimetric sensing system for the detection of carcinogenic aromatic amines. The probe showed high sensitivity and selectivity for aromatic amines over various competing soluble aromatic compounds and was capable for naked eyes or portable devices determination. The working mechanism was well studied through kinetic evaluation, experimental characterization and molecular dynamic simulations. This work does not only introduce a new graphene/protein hybrid material or a rapid and sensitive visualization of carcinogenic aromatic amines, but also spread the practical application of biomoleculegraphene interface strategy and further give a better understanding to the interaction of graphene and protein.

KEYWORDS. Layer by Layer Assembly, Graphene, Cytochrome C, Colorimetric Assay, Nanobio Interface


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Introduction Graphene oxide (GO) is one of the most widely used 2-D graphene derivatives due to its lowcost and facile fabrication in large scale, good water-solubility, convenience to modification, excellent electronic, thermal, mechanical and photophysical features and unique surface and chemical properties. GO has been applied in diverse areas including electronic devices1, photovoltaic2, hydrogen storage3, catalysis4, electrochemical5 and fluorescent6 sensors. Consisting of aromatic conjugated system and oxygen-rich hydroxyl and carboxyl groups, GO surface behaves either hydrophilic or hydrophobic at the same time. It is fundamental to understand the interaction between graphene and biomolecules such as DNA and proteins. For example, the unique interface condition of GO has been employed in designing fluorescent sensors, working by activating the “on-off” switch of GO-DNA interactions7-11. The interactions of GO and various proteins and peptides are also well studied. GO and other graphene derivatives have been used as immobilization matrix of enzymes for electrochemical12, electrochemiluminescent13, and catalytic applications14. Since GO is recently reported to be efficient artificial modulator of various enzymes, such as α-chymotrypsin15, serine protease16 and cytochrome c (cyt. c)17, it is promising to construct GO-based nano-vehicles and modulators through graphene-protein interaction. Very recently, Qu and his co-workers17 reported the effect on modulation of enzyme activity by GO. It is demonstrated in their work that cyt. c could be immobilized onto GO to form a cyt. c/GO composite by physical absorption and the peroxidase activity of cyt. c was highly improved in the composite. However, the cyt. c/GO composite by physical absorption is not very stable, that high temperature, change of pH, strong ionic strength, and metal ions such as Cu2+ and Mn2+ would be able to dissemble the cyt. c/GO composite or poison the enzyme


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activity of cyt. c. The low stability highly limited the potential applications of their cyt. c/GO composite. Enzyme-involved colorimetric sensors have been widely developed for various analytes determination and enzyme-linked immunosorbent assay (ELISA), due to their high sensitivity, applicability for biological sample and the convenience for visual detection. A typical enzymeinvolved colorimetric sensor included a redox enzyme, eg. horseradish peroxidase (HRP)18, and hemin-DNA complex19, and a substrate able to perform color-changing redox interactions, eg. 2, 2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) composite (ABTS) and 3, 3’, 5, 5’tetramethylbenzidine (TMB). However, ELISA kits are limited in a lot of conditions due to their high cost in fabrication and storage. The enzymes employed in ELISA are usually sensitive to environment. High temperature and microbiological contamination give rise to the risky of the disability of ELISA kits. Low temperature facilities (refrigerator, dry ice and liquid nitrogen) are often required for ELISA kits storage and delivery, which largely increase the cost and also limit their application. For most design of ELISA system, the enzymes are homogenesis in aqueous solution, which also hinders the recyclability of the expensive enzymes. Thus, there is a strong demand for increasing the stability, and also lowering the cost of the enzymes applied in ELISA. Layer-by-layer (LbL) assembly20 is a cheap and versatile method to construct high-quality thin films using two polyelectrolytes with opposite electrostatic charges. It has been confirmed in a lot of systems that LbL technique is an efficient way to embedding enzymes into thin films while remaining their bioactivity as well as strengthening the stability. Protein-involved LbL films showed great potential in opto-electrical devices21, biosensing22, tissue engineering23 and drug delivery24. Furthermore, the LbL assemblies were easy to be recycled that the LbL thin films could be continuously used for long time with high enzyme activity and recollected by simply


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mechanical separation. Thus, the LbL assembly is an ideal technique that would overcome the drawbacks of the current ELISA assays, such as low resistance to environmental change, high cost to store and deliver, difficulties to recycle the enzymes, and so on. Inspired by the previous work, we attempted LbL approaches to construct hybrid architectures of GO and cyt. c, achieving high enzyme activity where GO serves as an enhancer, and high stability against environment change (eg., temperature, pH, ionic strength, interfered metal ions) at the same time. Herein, the assemblies of cyt. c/GO hybrids were fabricated through a LbL method (Scheme 1). The structural morphology and properties of the LbL hybrids were well characterized using electron microscopy and other spectra techniques. Furthermore, it was found that the peroxidase activity of cyt. c was largely enhanced in the LbL cyt. c/GO assemblies, and its enzyme activity showed great resistance to external environment change and could be easily recycled by simply centrifuge and wash. The LbL cyt. c/GO hybrids were applied as a colorimetric sensor for visual detecting carcinogenetic aromatic amines. The probe works by regulating the peroxidase activity of LbL cyt. c/GO hybrids, response in catalyzed colorimetric change of ABTS and peroxide. The working mechanism of the colorimetric response were illustrated by kinetic study, experimental characterizations using atomic force microscopy (AFM), and theoretical simulations by the means of molecular dynamics (MD).


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Scheme 1. Schematic representation of the fabrication of layer-by-layer assemblies of cytochrome c and GO. Experimental Section Reagents and materials. Graphite powder (spectral grade) and other chemicals (analytical grade) were provided by Sinopharm Group Chemical Regent Co., Ltd. (Shanghai, China). All solvents and chemicals in this work were used without further purification unless stated. Doubledistilled water was used throughout the experiments. Instrumentation. The absorption spectra were recorded with microplate reader (infinite M200 pro, TECAN, Switzerland). JEM-3011 transmission electron microscope (TEM) (JEOL Ltd. Japan), Nova 200 Nanolab Scanning electron microscope (SEM) (FEI, U.S.A.) and BioScope atomic force microscope (AFM) (NanoScope IIIa SPM System, Digital Instruments, Inc., U.S.A) were used to study the morphology. The element analysis was performed by XPS (Kratos, Axis


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Ultra XPS). Rigaku Ultima IV XRD was used for XRD and SAXS characterizations. The circular dichroism (CD) was analyzed on J-815 CD spectroscopy (JASCO, USA). The Raman spectra were measured by Renishaw InVia Reflex Micro Raman Spectrometer (Renishaw, UK) while the FT-IR measurement were performed on Nicolet 6700 spectrometer (Nicolet, USA). The ζ-potential were measured by a Nano ZS Zetasizer (Malvern Instruments, UK). A Xiaomi 2A smartphone (Xiaomi, China) with an 8-million-pixel camera was used as a portable detecting platform. Synthesis of graphene oxide (GO). Graphene oxide was synthesized from graphite powders through a modified Hummers method25. Typically, 0.5 g of graphite, 0.5 g of NaNO3, and 23 mL of H2SO4 were mixed in an ice bath. 3 g of KMnO4 was carefully added into the mixture in 10 min. The suspension was transferred into 35 °C water bath and stirred for 2 h. 40 mL of water was then slowly added by a dropping funnel. The solution was refluxed for 30 min at 95 °C. 100 mL of water was continuously added. Subsequently, 3 mL of H2O2 (30%) was dropped in to change the color of the solution from dark brown to yellow. The resulting suspension was filtered with a 0.22 µm microporous membrane, and further washed with 200 mL HCl (1 M) and 200 mL deionized water for twice. The brown solid was vacuum-dried at 50 °C for 24 h to obtain GO. Fabrication of LbL cyt. c/GO hybrid. Firstly, isopycnic differential centrifugation was applied to obtain GO with narrower distribution in size26. The sample under 10000 rpm for two hours were used for successive reaction. After that, the GO sample was washed with DI water for three times and then diluted to 2 µg/mL. Then, 2 µM cyt. c were slowly dropped into the GO sample to obtain the cyt. c modified GO. The obtained cyt. c modified GO were separated through centrifugation under 10000 rpm for half an hour and then washed twice with DI water.


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Dispersion of 2 µg/mL GO was then added into the sample to form an LbL cycle. The LbL cyt. c/GO assemblies were fabricated after 3-20 times of repeating addition of GO and cyt. c solutions, respectively. Design of Colorimetric Assay. For aromatic amines determination, 0.5 mM ABTS, and 2 µg/mL LbL cyt.c/GO hybrids were incubated in 160 µL 2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid solution (HEPES, 10 mM, pH 7.4). Then 20 µL aromatic amine was added, and hereafter 20 µL H2O2 (60 mM). The absorption of the solution was collected using a microplate reader in 10 min. An Android App, namely ColorReader, was developed with MIT App Inventor 2 for photo analysis. The design of colorimetric was the same with that above. Photos were taken using a Xiaomi 2A smartphone (Android 4.1.1 JRO03L system) for colorimetric analysis with ColorReader. The RGB value of the photo was employed for quantitative analysis. Real Sample Detection. The real sample determination was carried out referring to GB 19601-2004 (China). Firstly, the clothes sample was cut into strips. Clothes strips (~2g) was submerged in 17 mL citrate buffer (0.06 M, pH=6) and incubated at 70 ˚C for 15 min. Then 3.0 mL sodium dithionite (200g/L) was added in and the solution was incubated at 70 °C for another 30 min, to completely reduce the dyes. The solution was adjusted to pH 8~9 using sodium carbonate (100 g/L). The final solution was extracted with 20 mL chloroform for three times. Two to three drops of acetic acid was added to improve the solubility of aromatic amines. At last, the chloroform solution was distilled to dry at 40 °C and dissolved in 10 mL water for determination.


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Molecular Dynamics (MD) Simulations. The MD simulations were performed with Forcite Plus module in Materials Studio. The classic Dreiding forcefield27 was applied for bonded and unbonded interactions in the models. The Coulombic force was calculated atom-based with a distance cutoff of 1.55 nm. NVT ensemble was used at the ambient temperature of 298K, controlled by the Nosé method28. A time step of 1 fs was employed while the total production time was set up to 1 ns. Results and Discussion LbL Cyt. c/GO Hybrid. GO and other graphene derivatives have been applied in LbL assembled thin films since the last two decades. It showed great potential in ultrastrong materials29, electrochemical sensors30 and biomedical devices31. Proteins, including bovine serum albumin32 (BSA), glucose oxidase, glucoamylase33, and cyt. c34 were embedded into the LbL films with GO by various groups. For instance, Xia and his group34 fabricated LbL composites of sulfonated graphene and cyt. c, using a one-step method simply mixing sulfonated graphene suspensions and cyt. c solutions. However, the sulfonated graphene applied in their work required complicated chemical modification where highly toxic chemicals such as hydrazine were applied, giving rise to the cost of the final product and also the harm to the environment. Moreover, Xia and his colleagues applied reduced graphene oxide (RGO) as the precursors to fabricate the LbL product. However, according to the recent studies17, RGO would show negative affect on the enzyme activity of cyt. c, which limits the activity enhancement of Xia’s work. In this work, a cycled method was applied to obtain the LbL assemblies. That is, cyt. c solution was firstly dropped into the GO suspension to get a cyt. c modified GO, which was collected by centrifugation and washed by purified water. After that, more GO was added to form a double layer


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assembly. The final product was prepared by repeating the above steps. This method is not only cheap and simple, but also environmental friendly using aqueous solutions under physiological pH, ionic strength, etc. which are biocompatible.

Figure 1. TEM images for single-layer GO (a), cyt. c modified GO (b), LbL cyt. c/GO hybrids after 3 LbL cycles (c) and the ones after 10 LbL cycles (d). SEM images for dispersed GO nanosheets (e), cyt. c modified GO (f), and LbL cyt. c/GO hybrids after 10 LbL cycles (g). SEM


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image of cross-section view (h) and high resolution SEM image at GO surface (i) for LbL cyt. c/GO hybrids after 10 LbL cycles. The LbL assembly works through the electrostatic affinity between the two reagents with opposite charges. In this case, it is GO that is highly negatively charged while cyt. c shows significant positive charges. Rich in large amount of oxygen-consisting functional groups such as carboxyl, carbonyl, hydroxyl and cycloxyl, GO showed highly negative charges at neutral pH. The measured ζ-potential of GO was -36.7 mV at pH 7.0. Cyt. c is a basic protein with a high isoelectric point of 10.7, which exhibits highly positive charges at neutral pH. After absorbing cyt. c, the ζ-potential of GO changed to 15.8 mV, made it possible for the next step electrostatic absorbing of negatively charged GO layer. TEM and SEM characterizations were applied to study the LbL assembly process. Figure 1a, e showed the morphology of the dispersed GO sheets applied in this work. It could be seen that the GO sheets were well dispersed in freestanding monolayers. The monolayer GO sheets showed very weak peak around 9.2˚ on XRD spectrum (Figure 2d), reflecting the good quality of monolayer GO sheets. The size of the GO sheets varied from 600 nm to a couple of micrometers. After addition of cyt. c, it could be seen in TEM and SEM in Figure 1b and f that the cyt. c modified GO had a relatively rough and wrinkled surface. It was because of the adhesion of cyt. c on GO that may lead to interlayer overlapping and slightly aggregation. For the 3 times LbL cycled assemblies, the architecture trends to be multilayers rather than well-dispersed monolayers. After 10 LbL cycles, more wrinkles and furrows could be found on cyt. c and GO assemblies from TEM imaging and the thickness of the assemblies kept arising. The assemblies were quasi-plate shaped showing obvious layered structures, with a diameter around 2-3 micrometers and the thickness 300 nm. The SEM images in cross-section view showed that the LbL cyt. c/GO hybrids contained approximately 120 graphene sheets. The numbers of layers were remarkably higher than the numbers of LbL cycles. It was because, for homogeneous LbL


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assemblies, multi-layers would be easy to be formed in a single LbL cycle. The high resolution SEM showed the LbL cyt. c/GO hybrids had highly rough surfaces due to the absorption of proteins.

Figure 2. FT-IR spectra (a), UV-vis absorption plots for GO, cyt. c and LbL cyt. c/GO hybrids (b). XPS survey spectra (c), XRD plots (d) for GO and LbL cyt. c/GO assemblies. CD spectra for 0.5 µM cyt. c, cyt. c with 2, 4, 6, 8, 10 µg/mL GO, and LbL cyt. c/GO hybrids (e). Raman spectra for GO and LbL cyt. c/GO hybrids (f). In order to further demonstrate the formation of the LbL assemblies, spectroscopic characterizations were applied for GO and the LbL cyt. c/GO hybrids. From the FT-IR spectra in Figure 2a we could know that the GO nanosheets showed a broad absorption band of carboxyl groups around 3300 cm-1, and another pair of bands of carbonyls at 1654 and 1756 cm-1. In the cyt. c/GO hybrids, besides the bands already exists in GO, a new peak appears at 1562 cm-1 corresponding with the amide II band (C-N stretch coupled with N-H bending mode) of cyt. c. Similarly, GO showed a broad band around 260 nm in UV-vis spectra (Figure 2b) while the LbL


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cyt. c/GO hybrids exhibited a new absorption band at 410 nm, corresponding to the resonance absorption of the hemin moiety of cyt. c. XPS element analysis was further applied to study the content of each component as well as the change in chemical states of GO and the protein after the formation of the LbL cyt. c/GO hybrids. Element quantification showed that the GO contained of 70.6% carbon and 29.4% oxygen and almost no nitrogen while the LbL cyt. c/GO hybrids had 70.9% carbon, 19.5% oxygen, 9.2% nitrogen and 0.42% of sulfur. Using the nitrogen content above, the mass ratio of cyt. c and GO is evaluated to be 1.38:1. High resolution XPS spectra (Figure S6, S7) showed that the chemical states for either carbon or oxygen did not change much after the formation of the LbL cyt. c/GO hybrids. The C 1s and O 1s XPS spectra for the LbL cyt. c/GO hybrids could be regarded as the linear combination of those for cyt. c and GO. It could be concluded that neither obvious oxidation nor reduction happened in the LbL assembly process that both cyt. c and GO keep their chemical states in the LbL hybrids. During the assembly process, there are usually conformation change for the proteins embedding into other materials. CD spectra were employed to illustrate the possible conformation difference between free cyt. c in aqueous condition and the embedded ones in the LbL cyt. c/GO hybrids. Through titration plots of GO to cyt. c, it was observed that the negative CD peaks at 222 nm and 209 nm both decreased as more GO were added into the cyt. c aqueous solution, corresponding to the dissembles of α-helices in the protein. Meanwhile, a new positive peak appeared at 255 nm when GO added into cyt. c. This peak corresponded to the absorption of phenylalanine residues from cyt. c. It is known that phenylalanine is a typical hydrophobic amino acid containing aromatic rings. We proposed that the rise of the 255 nm CD (Figure 2e) is attributed to the π-π stacking of the aromatic rings from phenylalanine residues to the aromatic


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conjugation surfaces of GO. More interestingly, the LbL cyt. c/GO hybrids did not show any obvious CD signal across the range from 190 nm to 400 nm. It implies that the conformation of cyt. c embedded in the LbL cyt. c/GO hybrids is probably different from either free cyt. c in aqueous solution or those physically absorbed on GO surface. All in all, the conformation of cyt. c changed a lot after assemblies onto GO from the CD characterizations. XRD together with Raman spectra were used to study the structural change in the LbL assembly. Different with GO which showed a weak XRD peak at 9.2˚, the LbL cyt. c/GO hybrids showed two peaks at 9.8 and 8.2˚, corresponding to distances of 0.90 and 1.08 nm. It implied that the modification of cyt. c might influence the average distance between graphene sheets, which is possibly induced by the change of surface electrostatic charges of GO. SAXS (Figure S5) provided more detailed information on larger scale architectures. The LbL cyt. c/GO hybrids showed a peak around 1.8˚, corresponding to a relatively large distance of 4.9 nm. The increased distance is comparable to the size of cyt. c, offered distinct evidence of the implantation of cyt. c into the gaps between GO layers. Through Raman spectroscopy (Figure 2f), no obvious shift was observed for D and G bands and the intensity ratios of D band and G band were calculated to be 0.88 and 0.87 for GO and LbL cyt. c/GO hybrids, respectively. It can be inferred that the defects and oxidation states of GO did not change much after the formation of LbL hybrids35. However, the LbL cyt. c/GO hybrids showed a lower intensity ratio of 2D1 over 2D2 band than that for GO. The enhancement of the ratio is probably caused by the increase of the numbers of layers, which is already confirmed by TEM and SEM imaging.


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Figure 3. Photographs of GO and LbL assembled cyt. c/GO (a). Relative enzyme activities of GO, cyt. c modified GO and LbL cyt. c/GO hybrids at ambient condition, after 10 min boiling, in the presence of 10 mM sodium chloride, and ethanol, respectively. Normalized relative enzyme activities of GO, cyt. c modified GO and LbL cyt. c/GO hybrids at various pH value. Recyclability of cyt. c modified GO and LbL cyt. c/GO hybrids. After the formation of the LbL assembled cyt. c/GO hybrid, the enzyme activity greatly enhanced comparing to free cyt. c. Containing equal quantity of cyt. c, the LbL cyt. c/GO hybrids showed 3 times higher activity than cyt. c modified GO and 22 times than free cyt. c in solution (Figure 3b). The LbL cyt. c/GO hybrids showed remarkably higher resistance to environment change than regular protein modified nanomaterial composites, which are usually sensitive to pH, temperature and solvents. It is convinced that LbL method is an ideal strategy to stabilize physically absorbed protein-nanomaterial composites. In Figure 3b it showed that the LbL cyt. c/GO hybrids is resistant in enzyme activity after 10 min boiling, 10 mM salt treatment or ethanol emersion. As a peroxidase, the cyt. c could be easily deactivated by high temperature boiling over 95˚ C. Ethanol is reported36 to be able to strongly change the conformation of cyt. c.


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which also resulting in enzyme deactivation. High ionic strength would reduce the electrostatic interaction in aqueous solution. That’s why the enzyme activity of cyt. c modified GO would decrease in the presence of 10 mM sodium chloride as the ionic strength dissembled the protein modification through electrostatic affinity. However, the LbL cyt. c/GO hybrids were affectless to the above interferences as the protein was strongly immobilized in the LbL assemblies. Furthermore, the assemblies held high activity in a wide range of pH value from 4 to 9 while free cyt. c and its modified GO showed diverse activity in such range (Figure 3c). Moreover, the LbL cyt. c/GO hybrids can be reused using simple centrifugation (600 rpm, 10 min) and dissolve in water again. They held high activity after recycled for more than 5 times without any obvious activity loss whereas the cyt. c modified GO was not able to overcome more than 3 times recycling (Figure 3d). All in all, the resistance to extreme environment change and the practicality for recycling uses for LbL cyt. c/GO hybrids largely improved comparing to free cyt. c and cyt. c modified GO. Those advantages mentioned above made it possible for LbL cyt. c/GO hybrids to be applied in practical devices41 such as colorimetric sensors. Application for Colorimetric Detection of Carcinogenic Aromatic Amines. Aromatic amines are highly hazardous compounds which are toxic to humans and aquatic life37. Discharge of aromatic amines to environment would bring serious environmental pollution and terrible health risk. Long-term contact with aromatic amines even in a small amount would lead to chronic poisoning and malignant tumors. In early 2013, the “toxic” school uniforms in Shanghai have greatly drawn the social focus. Carcinogenic aromatic amines (CAAs) were detected in school uniforms, which were provided to 21 fundamental schools. And more than 20 thousand students were involved. Carcinogenic aromatic amines in clothes are generated from azo dyes, the so-called “banned aromatic amine dyes”, which are strictly limited in clothes and skin-


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contacting products across EU, USA and China. For instance, the directive 2002/61/EC of the EU Parliament and of the Council has listed 24 aromatic amines not to be allowed in textiles and feather articles, in detectable concentration of 0.15 µM (30 ppm). However, the current analytical techniques for aromatic amines in textiles and feather products, including gas chromatography/mass spectroscopy (GC/MS)38 and high performance liquid chromatography (HPLC)39, all require complex and time-consuming pre-treatment before determination. The instruments employed in such detecting methods are expensive and complicated. Thus, there is an urgent demand for rapid and facile method for on-site and real-time aromatic amines detection. β-Naphthylamine (NA) and 4,4’-diaminobiphenyl (DABP) are the most toxic compounds among the 24 carcinogenic aromatic amines listed by EU Parliament and of the Council. Both NA and DABP are classified in Group 1 (carcinogenic to humans) by International Agency for Research on Cancer (IARC). Thus, NA, DABP and aniline (BA), the simplest aromatic amine, were utilized to test the applicability of the colorimetric sensor. In order to verify the selectivity for aromatic amines, other competing aromatic compounds including phenol (PO), 2-nitrophenol (2-NP), 3-nitrophenol (3-NP), 4-nitrophenol (4-NP), 4-cholrophenol (4-CP), salicylic acid (SA), phthalic acid (4-PA), 4-toluenesulfonic acid (4-TSA), 1-naphthalenecarboxylic acid (NCA) and 1-naphthaleneacetic acid (NAA) were tested (Figure 4). The colorimetric sensor was adopted in aqueous solution, thus only water soluble aromatic compounds were considered as interferences. It was found that only aromatic amines could inhibit the enzymatic activity of the LbL cyt. c/GO hybrids. Among the three aromatic amines employed, NA was the strongest inhibitor while BA was the weakest. It can be understood that NA has the largest aromatic conjugation to stack on GO surface yet BA has the smallest. As for DABP, though it has two aromatic rings, the two


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benzene rings are not coplanar. The intersection angle of the benzene rings in DABP is as big as 44˚, which limits the stacking interaction of DABP with GO. That is why the inhibiting effect of DABP was between those of NA and BA. Aromatic amines are distinguished from those aromatic compounds because aromatic amines showed stronger π−π stacking interaction on GO, which would possibly disintegrate cyt. c/GO hybrids or poison the enzyme, resulting in weak enzymatic activity. In addition, electrostatic interaction is proposed to be essential to the selectivity. Enrich with hydroxyl and carboxyl groups, GO is negatively charged at physiological pH. Aromatic amines are weakly alkaline. They are positively charged in aqueous solution and would be electrostatically attracted to GO. Similarly, Coulomb repulsion could sufficiently explain NCA and NAA consisting two aromatic rings would not inhibit the activity of the LbL cyt. c/GO hybrids. The colorimetric assay for aromatic amines was quite sensitive. For NA detection, the probe showed good linear color response from 5 nM to 3 µM (Figure 4). The detection limit for NA with naked eyes was around 1 µM. As far as our knowledge, it is the most sensitive method for visual determination of carcinogenic aromatic amines without using any equipment. Applying microplate reader (infinite M200 pro, TECAN, Switzerland) for visible absorption signal readout, the limit of detection (LOD) was as low as 1 nM. Moreover, the visualization of analytes was very fast. The colorimetric response could be read out in ten minutes, which built an avenue to detection on site and emergency program40. Overall, the optical carcinogenic aromatic amine probe was appropriate for practical sample determination. The assay was applied in clothes samples referring to GB 19601-2004, P. R. China (Experiment Details, ESI). The assay showed high recovery in real sample determination (Table S1). Thus, the sensing system is a potential tool for rapid and sensitive monitoring of carcinogenic aromatic amine in textiles and dresses.


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Figure 4. (a) Colorimetric response of ABTS and LbL cyt. c/GO hybrids with H2O2 and NA input. (b) Linear plot of absorption at 417 nm to NA concentration. (c) Time curve of absorption at 417 nm of cyt. c with GO and NA (5 µM). (Empty square –cyt. c and empty circle –cyt. c-NA without GO; solid square –cyt. c and solid circle –NA with LbL cyt. c/GO.) (d) Time curve of absorption at 417 nm in the presence of NA of various concentrations. (e) Colorimetric response of ABTS/H2O2 and LbL cyt. c/GO hybrids in the presence of aromatic amines and other competing soluble aromatic compounds. The concentration for ABTS, H2O2, cyt. c, GO, aromatic amines and other aromatic compounds was 0.5 mM, 6 mM, 0.5 µM, 2 µg/mL, 5 µM and 50 µM, respectively.


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A commercial smartphone was applied as a portable detecting device, and a specialized Android App was developed (Figure S12). RGB values were used for quantitative detection. Green channel was found to be the most sensitive, thus G value was applied in the following research. Using smartphone as portable platform, the probe for carcinogenic aromatic amines exhibited rather high sensitivity. For a well-trained user, the portable detection platform showed a wide detecting range from 10 nM to 20 µM. The LOD for NA of the portable device was comparable to that of a professional UV-vis microplate reader and was remarkably higher than naked eyes. The colorimetric sensor was quite practical for on-site determination and would do a favor to quality control in manufacture of clothes and textiles. Kinetics Process, Working Mechanism and MD Simulations. The kinetic process of the color changing interaction was studied (Figure 4c and d). It was observed that the colorless substrate ABTS was oxidized by H2O2, catalyzing by cyt. c. It was notable that free cyt. c did not show high performance in CAA detection. The enzymatic activity of cyt. c was much weaker and showed no obviously colorimetric response for aromatic amine. Whereas in the case of LbL cyt. c/GO hybrids, the absorption of ABTS• at 417 nm dramatically increased after H2O2 addition The plot of absorption (A) at 417 nm to time (t) fits well to Michaelis-Menton function, ‫ = ܣ‬3.019‫ݐ‬/(14.57 + ‫)ݐ‬

(1)

As for the LbL cyt. c/GO hybrids in the presence of aromatic amines, however, the absorption at 417 nm stays low for a period of time. Then the absorption rises rapidly after exceeding a threshold value. All the absorption reaches a same maximum in 2 hours. The time for 50% of maximal effect (ET50) is proportional to the concentration aromatic amine (c), ‫ܶܧ‬50 = 0.1184ܿ − 1.031

(2)


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The kinetic process is quite coincident with the mechanism we proposed. Aromatic amines inhibit the enzymatic activity by disintegrating cyt. c/GO hybrids through π−π stacking on GO surface. Aromatic amines are possibly consumed, leading to the final color change. The consumption of aromatic amines is probably due to the oxidation by H2O2, producing phenols and quinones. Inferring from kinetics, both the assembly of aromatic amines on GO and its consumption are zero-order reactions. It implies that all the reactions take place on GO surface. Since the enzymatic activity of the LbL cyt. c/GO hybrids was recovered after the reaction, simple centrifugation (11000 rpm, 10 min) could be used to separate LbL cyt. c/GO hybrids for recycling.


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Figure 5. (a). Proposed working mechanism of the colorimetric response of CAAs using LbL cyt. c/GO. Highlighted hemin moieties in the equilibrium structures for free cyt. c (c) and cyt. c absorbed on GO (d).Final conformation structures of cyt. c (d), GO sheet (e), cyt. c absorbed on GO (f), NA molecules absorbed on GO surfaces (g), cyt. c absorbed on GO in the presence of NA molecules (h) from MD simulations. Though a lot of researchers have observed the enzyme activity enhancement after the protein embedded to nanomaterials such as graphene, the mechanism of the activity enhancement have not been well discussed for long time. Xia simply attributed the activity enhancement of cyt. c to the confinement effect in his LbL work34. Qu and his coworkers noticed the conformation change in the cyt. c modified GO and reduce GO from CD spectroscopy but more detailed information of the conformation change is not provided in their report17. In this work, a working mechanism was proposed in the help of MD simulations combining with experimental characterizations (Figure 5). The structure of cyt. c was imported on the basis of the crystallographic data from the protein database bank (PDB database, ID: 3cyt). A diamond-shaped graphene sheet with a side length of 5.6 nm were applied as a simplified model in the simulations. All the dangling bonds at the edge of the graphene sheets were saturated with hydroxyls groups to mimic oxygen-rich GO. From the experimental results, we know that although the performances of cyt. c modified GO and the LbL cyt. c/GO hybrids are slightly different, but they showed more things such as the same response, similar selectivity, etc., in common for the detection of CAAs. We proposed that for cyt. c modified GO and the LbL cyt. c/GO hybrids, the working mechanism of CAAs sensing should be similar. Thus, we applied cyt. c modified GO to clarify the model as well as lower the computational cost in this work. From the simulation results, it was clearly shown that a composite of GO sheet and cyt. c was formed after MD equilibration. In the composite, the cyt. c unfolded and spread itself to the


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GO surface, with dissembling of α-helices. It was noticed that the phenylalanine residues showed high affinity to GO surface. The average distance of the attached phenylalanine residues to GO sheet was measured to be 3.2 Å, with is the typical distance for the π-π stacking of aromatic rings. Those results are in surprisingly high coincidence with our results of CD measurements represented in the above sections. It is known that the peroxidase activity of cyt. c comes from its hemin moiety. The structural configurations around the hemin moiety for free cyt. c and the cyt. c/GO composite were illustrated (Figure 5d, e). Comparison to that for free cyt. c, the hemin part in the cyt. c/GO composite showed a distorted porphyrin-iron plane and the hemin center was exposed due to the unfolding of the protein. We suggested that the distortion of the porphyriniron plane might lead to a conformation which is possibly easier for peroxide catalysis with a lower energy barrier. The expose of the hemin moiety is also beneficial to the absorption of peroxide molecules to the catalytic center because of the lower steric hindrance. The distance of the iron center to GO surface is 1.30 nm, which is an efficient distance for charge transfer between them. The efficient charge transfer is also an important factor for the enzyme activity enhancement as GO is an electron abundant nanosheet. It is the first time to give the answer to the question why the cyt. c protein would show higher enzyme activity after conjugation to graphene from MD level. The interactions of NA molecules and free GO sheets are studied. Consisting of aromatic rings, NA molecules showed strong adherence to GO surface in either single layer absorption when the amount of NAs was small or multiple layer absorption when large amount of NA molecules were included (Figure 5g). The absorption of cyt. c onto GO surface in the presence of NA molecules is also simulated. It can be seen in Figure 5h that with a lot of NA molecules, the GO sheets tends to bend like a bow to bind up the cyt. c. A layer of NA molecules were


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intersected between the cyt. c and GO, making their distance much larger. There are a large number of NA molecules surrounded the protein, close to the sites of hydrophobic and aromatic residues of amino acids. Based on the MD results, we suggested that three factors would contribute to the inhibition of NA to the enzyme activity of cyt. c/GO hybrid. Firstly, the existence of NA molecules lowered the absorption energy of cyt. c to GO. In the absence of NA molecules, the absorption energy to cyt. c protein is calculated to be 48.9 kcal/mol whereas the one with NA molecules decreased to 36.7 kcal/mol. It is demonstrated that there are competition of NA and cyt. c in the absorption process which may lead to the dissemble of cyt. c/GO hybrids. Secondly, in the presence of NA, a layer of NA molecules were embedded between the cyt. c and GO, making the distance larger for the iron center of hemin moiety to GO surface (2.0 nm), which hindered the electron transfer between them. At last, in the presence of NA molecules, the conformation of cyt. c changed a lot and there were a large amount of NA molecules absorbed onto the protein. The conformation change as well as surface absorption of NA molecules may both result in catalytic poisoning of the enzyme. In order to further verify the mechanism we assumed, atomic force microscopy (AFM) was utilized. Since it was difficult to observe the enzyme embedded in the LbL assemblies, the cyt. c modified GO were applied to verify the mechanism as a proof-of-concept, corresponding to our MD simulations. One can clearly see in Figure S1a that the naked GO shows highly smooth surface where the height differential on the GO layer is smaller than 0.2 nm. However, in the presence of NA, the roughness of the GO sheet remarkably increases with a higher surface height differential of 0.4 nm, which corresponds to the thickness of NA molecule. It was directly observed that cyt. c was immobilized on GO in Figure S1b. The thickness of cyt. c/GO hybrids are remarkably higher than free standing GO sheets (typically 0.4 nm). However, in the presence


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of aromatic amines, cyt. c and GO separated from each other (Figure S1e). GO sheet was positioned from AFM, which was much thinner than cyt. c/GO hybrids. It should be noted that the cyt.c modified GO with NA is also rougher than free GO. As to the Lbl cyt, c/GO hybrids, large assemblies of proteins can be found on the surface of the composites (Figure S1c). But in the presence of NA, no protein assemblies were observed on the composite surface. The LbL cyt.c/GO composites with NA shows a similar roughness to the free GO with NA existence. The roughness of GO sheet with aromatic amines confirms the π-π stacking assemblies of aromatic amines on GO surface, which gives convinced evidence to the mechanism we proposed.

Conclusions In summary, a new LbL assembled structure of GO and enzyme was fabricated and a rapid and sensitive assay for colorimetric determination of carcinogenic aromatic amines was designed using the LbL assemblies. This work showed a great example to maximize the benefits of LbL assemblies of bio-nano composites in practical applications. The assay worked by regulating the enzymatic activity of the LbL cyt. c/GO hybrids through interface-based reaction. The assay showed good specificity for aromatic amines against various aromatic compounds in aqueous solution. The kinetics and mechanism of the probe were well discussed combining experimental characterizations and MD simulations. It was proposed that aromatic amines strongly inhibit the enzymatic activity of cyt. c/GO hybrids through π−π stacking on GO to construct a passivating surface. The carcinogenic aromatic amine sensor is quite sensitive and do not require any expensive equipment, and the sensor is practical for real sample determination. It is one of the most sensitive sensing method for carcinogenic aromatic amine with naked eyes. This work would not only propose a design of a colorimetric probe or a new LbL assembly, but also offer a


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new view of modulating enzymatic activity by molecular assemblies with nanomaterial and better understanding of the interaction of protein and graphene. The capacity to be applied in portable devices of the sensor also showed great potential in on-site determination and quality control, especially for clothes manufacturing and trading.

ACKNOWLEDGMENT. This work was supported by the National Natural Science Foundation of China (No. 21675053, 21635003, 21775044). Supporting Information Available: Supporting ﬁgures and tables.


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SYNOPSIS TOC.


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Colorimetric Detection of Carcinogenic Aromatic Amine Using Layer

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