Measurement of Luminescence Intensity on Textiles Using Luminous Bacterial Biocatalytic System

Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

Measurement of luminescence intensity on textiles using Luminous bacterial biocatalytic system

Sweta IYER1,2, N BEHARY1,2, V NIERSTRASZ 3 1*ENSAIT-GEMTEX, F-59100, Roubaix, France 2 Université Lille Nord de France, F-59000, Lille, France 3 Textile Materials Technology, Department of Textile Technology, Faculty of Textiles, Engineering and Business, University of Boras, SE-50190, Borås, Sweden

ABSTRACT Nature is the most exquisite thing around us with the existence of living organisms exhibiting different phenomena such as water repellency, touch sensitive plant and chameleon skin. Some of these phenomena inspired scientists to explore and design smart fabrics biomimicking the behaviour or pattern in living organisms. Bioluminescence is one such phenomenon where-in different living organisms such as firefly, jelly fish and crustaceans have the ability to impart visible light of specific wavelength, by enzyme catalysed reactions. Existence and study of such light emitting living organisms have been carried out, and harnessing these reactions has already transformed significant areas of medical field and clinical diagnosis, but research work on transforming this into living light is limited. In the present study, luminous bacterial system was investigated to assess and detect the bioluminescence behaviour onto the textile material. In the Luminous bacterial system, in vivo biochemical mechanism involves two different enzymes as well as different substrate components. Emission of light due to in vivo luminous bacterial reaction mechanism is seen in visible region. For in vitro reaction mechanism study, physical adsorption technique was used to graft both enzymes on plasma activated PET nonwoven textile and when substrates were introduced manually during the analysis, the biochemical reaction leading to light production occured. A Luminometer equipment was used to determine the light intensity in terms of Relative light units (RLU). The measurement results were obtained for nonwoven plasma treated PET with enzyme and substrate addition at different concentration and RLU value was obtained. The analysis data revealed that light intensity in RLU could be recorded by introducing both the enzymes and substrates on textile material, however intensive research is required in order to observe emitted light through the naked eye. The research study will help to attain new approaches for obtaining luminescent textiles using biobased resources.

KEYWORDS Biomimicking, Luminous bacterial, Luminescent textile.

Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

INTRODUCTION Nature has designed a few bio-based molecules which are responsible for bioluminescence in some living species. There are diverse living organisms present in sea as well as land environment such as fireflies, jellyfish, fungi and bacteria that produce light via enzymatically catalysed chemical reactions. Many research works have been carried to study bioluminescence, by considering the morphology, physiology and chemistry involved in light producing organisms. The molecules responsible for bioluminescence in jelly fish was discovered by Nobel Laureate ‘Osamu Shimomura’ and different aspects of bioluminescence chemistry have been reviewed [1]. The light emitting phenomenon can be seen in luminous organisms due to two principal components luciferin (the substrate) and luciferase (the enzyme). `Firefly´ is one such organism of luciferin-luciferase reaction system, wherein presence of specific enzyme (firefly luciferase) oxidizes corresponding substrate (generically known as luciferin) in presence of oxygen and other co-factors such as ATP and magnesium ions to emit light (Fig. 1).

ATP O2

D- Luciferin

Firefly luciferase + Mg2+

Light PPi CO2 AMP

Oxyluciferin

Figure 1: Reaction mechanism of Firefly (luciferin-luciferase)

Depending upon the ecological presence and physiology behaviour of different luminous organisms, the excitation mechanism and energitics of bioluminescence vary. Hence, there exist a broad color spectrum of bioluminescent signal from blue, green and red shifted emissions ranging from 450 to 580 nm [2]. Bioluminescence has wide application in the field of bioresearch. Although bioluminescence reaction systems have been extensively used as environmental biosensors for Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

screening and evaluating the biological effects of toxicity [3], bioluminescence assay to determine low level of microbial contamination on textile material [4], luminescence in yeast containing bacterial luciferase for screening or detection of antifungal compounds [5] but until now the biobased reaction system has not been studied to produce bioluminescent textiles. Applications of luminescent textiles for architecture, safety, sportswear and apparel [6] are wide but there are also other applications, which exist such as wearable sensors and biosensors [7] [8][9]. Hence, it is of great interest to create different application based on the bioluminescence system existing in nature. In the current paper, the application approach will allow developing the concept regarding the mechanisms of interaction of substrates with the light emitting proteins or enzymes on the surface of textile material. In particular, the in vivo luminous bacterial system involves biochemical reaction consisting of specific enzyme and substrate components [10]. The in vitro reaction studied involved physical adsorption of luciferase enzyme on textile support and substrates introduced manually to count the photons of light in RLU using luminometer. Thus, the present work focuses on designing of biobased luminescent textile.

EXPERIMENTAL

Materials and Methods

The enzymes and substrates used in the experiments were purchased commercially. The measurements were performed using a Luminometer MicrolumatPlus LB 96V (France). The equipment detector consist of ultra fast single photon counter system having spectral sensitivity range for wavelength of 380 – 630 nm. The light emission was measured as Relative light units (RLU).

Luminometer equipment

A luminometer equipment is used to measure the light produced due the chemical reaction occurring and can be measured in the form of Relative light units (RLU). Generally, it is used to measure chemical marker ATP, which is a compound found in all types of plant, wherein the amount of light is proportional to the amount of ATP present [11].

Nonwoven PET sample

A 100 % nonwoven polyester (PET) fabric of density 230 g/m2, diameter of 12 µm and thickness of 950 µm was used. The fabric was initially cleaned using Petroleum ether in Soxhlet followed by rinsing and drying of the fabric in ethanol and distilled water subsequently to remove the spinning oil and surface impurities [12]. Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

Plasma treatment of nonwoven PET

Plasma treatment modifies the polymer matrix using plasma gases. The plasma gases made up of mixture of free radicals, electrons and ions activate the PET fiber surface [13]. Hence, the nonwoven PET was plasma treated for enzyme immobilization experimental study. A `50 x 50 cm´ PET nonwoven fabric was cut into square pieces according to the electrode length of the plasma machine. The plasma treatment was performed using an air atmospheric plasma machine called ¨Coating Star¨ manufactured by Ahlbrandt system (Germany). The following machine parameters were kept constant; the electrical power of 1kW, frequency of 26 kHz, speed of the fabric 2m/min, electrode length of 0.5m, and an interelectrode distance of 1.5mm along with treatment power of 45 kJ/m2. After plasma treatment, each treated sample was kept in aluminum foil well away from light.

Contact angle and capillarity mesurement methods

The plasma treated samples were characterized using contact angle and capillarity measurements on a tensiometer to quantify the surface treatment modifications. Using the apparatus 3S Balance from GBX Instruments (France) the capillarity weight (g) of the meniscus (Wm) of fabric sample at 3 min was measured using Wilhelmy principle method [14]. The water contact angle ( ) at the nonwoven sample surface could be determined from the calculated meniscus weight (Wm) using𝜃𝜃 below equation (1), [15]. x = × × (1)

-1 As both the ( ) surface tension𝑊𝑊𝑊𝑊 of 𝑔𝑔liquid𝛾𝛾 𝐿𝐿water𝑐𝑐𝑐𝑐𝑐𝑐 in 𝑐𝑐` mN m𝑝𝑝 ´and ( ) perimeter of the contacting surfaces in `mm´ were𝛾𝛾𝐿𝐿 known. The increase in surface energy of plasma treated𝑝𝑝 sample can be seen by a reduction in water contact angle. The contact angle of plasma treated PET obtained with value 0° in comparison to the untreated PET fabric with value 141°.

Enzyme immobilization for PET activated surface after plasma treatment

Immobilization on PET nonwoven textile was carried out using direct sorption/ adsorption technique. A `1 x 1 cm´ of plasma treated PET nonwoven fabric was cut and placed in a petri dish with enzyme luciferase and FMN reductase enzyme involved in reaction of luminous bacterial system. The petri dish was stored in a refrigerator at 4 ֯ C for 18 hours. Further, the nonwoven PET was removed from the petri dish and then dried at room temperature. Meanwhile the substrate components of luminous bacterial reaction system were freshly prepared. The immobilized textile sample was introduced in the sample holder of the luminometer equipment and the freshly prepared substrate components were injected manually to determine the intensity of the reaction in the form of Relative Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

light units (RLU) (as seen in Fig. 2).

Substrate interaction Enzyme A with enzyme

Immobilization֯ Injected manually

Textile Textile Textile

Substrate components

Measurement of RLU Luminometer

Figure 2. Schmatic representation for preparation and flow diagram for luminescence analysis on textiles

RESULTS AND DISCUSSIONS

Measurement of reaction mixture using luminometer

At the first stage of analysis, the reaction mixture was studied in the solution form and intensity was measured using luminometer. Different concentration of luciferase enzyme and substrate components were prepared. Optimization of the concentration for enzyme and substrate components provided intensity (RLU) values and maximum intensity was attained at about 9000 RLU. The measurement were repeated and was noted in the time frame of 2 minutes after manually injecting the substrate components required for the reaction to occur. As seen in Figure 3. the RLU value was obtained in the range of 8000 to 9000 for three different measurements. Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

Figure 3. Relative light units (RLU) observed for solution mixture of luminous bacterial system

At the next stage of analysis, the physically adsorbed enzyme immobilized textile was measured for luminescence intensity. Plasma treatment modifies the surface properties by inserting chemical functionalities of free radical onto supporting material allowing textile surface for grafting of enzymes or proteins, enabling easy diffusion[16]. There exist different methodologies to immobilize the enzyme on plasma acivated nonwoven such as entrapment and direct sorption, however, in this research work the direct sorption technique has been used to determine the intensity on textiles using luminous bacterial system [13]. The intensity measurement for the immobilized textile in presence of the substrate components provided intensity value of about 500 RLU. The repeated analysis measurement provided the intensity value in the range of 450 – 500 RLU. It was found experimentally that analysis of enzyme treated nonwoven textile using luminometer in presence of luminous bacterial system led to photons of light and the RLU value could be determined as seen in Fig. 4.

Figure 4. Relative light units (RLU) observed for immobilized textile of luminous bacterial system Textile Science and Economy 2019 French - Croatian Forum rd th January 23 to 24 , 2019, Zagreb, Croatia

The bioluminescence phenomenon occuring in bacterial luciferase exhibit emission of visible light due to chemical reaction at wavelength 470 nm [17]. The luminous bacterial biocatalytic system using luminous bacteria is abundantly been used to develop specific methods of analysis of a variety of metabolites and for determination of enzyme activity; it serves as test device for determining the integral toxicity of different environments. Normally, application of luminous bacteria system have been explored for use of environmental biosensors and bioassays based on reaction system [3] [18]. However, in the current study the luminous bacteria reaction system have been introduced on textile surface and the maximum intensity achieved as relative light units (RLU) is measured and discussed.

CONCLUSIONS

Hence, the results obtained in this work demonstrated the possibility of fabricating a luminescence textile surface consiting of luminous bacteria reaction system. Fixation of enzymes on textile surface can be achieved further using covalent bonding with chemical reactants and biological protein-protein interaction. The entrapment of enzymes in natural polymer gels such as gelatin and starch improves the enzyme stability and the verification of the possibities and potential of these method is subject for further evaluation to achieve stable enzymatic system and increase the RLU value on textiles.

Acknowledgements

This research project is financially aided by European commission, Erasmus Mundus joint Doctorate program with Specific Grant agreement n° 2016-1353/001-001-EMJD under the framework of Sustainable management and Design for Textiles (SMDTex).

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