Realization of Beeswax-based Fabric Microfluidic Channels using PVC Clear Sheet Stencils

doi:10.21203/rs.3.rs-3864102/v1

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Realization of Beeswax-based Fabric Microfluidic Channels using PVC Clear Sheet Stencils

https://doi.org/10.21203/rs.3.rs-3864102/v1

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Microfluidic channels fabricated over fabrics or papers have the potential to find substantial application in the next generation of wearable healthcare monitoring systems. The present work focuses on the fabrication procedures that can be used to obtain practically realizable fabric-based microfluidic channels (µFADs) utilizing patterning mask and wax, unlike conventional printing technique. In this study, comparative analysis was used to differentiate channels obtained using different masking tools for channel patterning as well as different wax materials as the hydrophobic barriers. Drawbacks of the conventional tape and candle wax technique was noted and a novel approach was used to create microfluidic channels through facile and simple masking technique using PVC clear sheets as channel stencils and beeswax as channel barriers. The resulting fabric based microfluidic channels with varying widths as well as complex microchannel, microwell, and micromixer designs were investigated and a minimum channel width resolution of 500 µm was successfully obtained over cotton based fabrics. Thereafter, the PVC clear sheet-beeswax based microwells were successfully tested to confine various organic and inorganic samples indicating vivid applicability of the technique. Finally, the microwells were used to make simple and facile colorimetric assay for glucose detection and demonstrated effective detection of glucose levels from 10 mM to 50 mM with significant color variation using potassium iodide as the coloring agent. The above findings clearly suggests the potential of this alternative technique in making low-cost and practically realizable fabric based diagnostic device (µFADs) in contrast to the other approaches that are currently in use.

fabric based microchannels

microfluidic fabric based analytical devices (µFADs)

beeswax

colorimetric detection

wearable devices

The need for fast and precise detection or manipulation of biological or biochemical entities, with a focus on high sensitivity and accuracy, has driven the advancement of BioMEMS and microfluidics-based miniaturized analytical devices. These devices have found diverse applications, including drug delivery, disease screening, and sensor deployment, catering to the growing demand for efficient and effective biomedical technologies[1, 2]. Microfluidic channels are employed to control and engineer the flow of fluids or suspended particles across small surfaces. These channels are designed to ensure that surface forces play a dominant role over volumetric forces, especially in channels with dimensions on the scale of a few tens of micrometers. This enables precise manipulation and regulation of fluid behavior within microscale environments, offering a wide range of applications [3]. Several materials have been extensively studied and utilized in the fabrication of microfluidic channels, including silicon, glass, and various polymers such as PDMS, SU8, and paper[4]. These materials offer different properties and characteristics that make them suitable for development through different fabrication techniques as well as for various applications in the field of microfluidics. Silicon and glass provide excellent optical transparency and compatibility with microfabrication processes [5], while polymers like PDMS offer flexibility, ease of fabrication, and biocompatibility[6]. Paper-based microfluidic devices are advantageous for their low cost, disposability, self-transport of fluids and simplicity of use, making them particularly suitable for point-of-care diagnostics and resource-limited settings[7]. The choice of material depends on the specific requirements of the microfluidic application, such as biocompatibility, optical transparency, mechanical properties, ease of fluid transport, cost-effectiveness, etc[8–10]. Among these, PDMS (Polydimethylsiloxane) based microfluidic devices realized through soft lithography techniques have been most popularly used for the development of analytical devices [6, 8]. However, the requirement of specific master templates for any changes in design, spontaneous transport of fluids without the need for an external pumping system, and the need for a better wearable and disposable diagnostic device resulted in exploring papers as an alternative microfluidic platform. This demand led to the development of microfluidic paper based analytical devices (µPADs)[10–13]. Recently, microfluidic channels have also been realized over fabric based materials and are called as microfluidic fabric-based analytical devices (µFADs) or microfluidic cloth-based analytical devices (µCADs)[14, 15]. The inherent advantages of cloth or fabric are that they are economical, flexible yet highly durable, and portable with the ability to transport fluid by capillary action. These properties have made it a very attractive alternative for designing analytical devices for the detection of food adulterants, point-of-care diagnostic devices, wearable sensors, etc since the last decade [14]. P Bhandari et al. in 2011 developed a fabric based microfluidic platform by weaving wetting and non-wetting silk yarns to develop hydrophilic and hydrophobic regions [16]. Similar weaving technology was also used by Vatansever et al. in 2012 [17] to develop textile based microfluidic channels having pH sensitive channels which were able to switch water based solutions in different directions depending upon the pH of the channel yarn.A. Nilghaz et al in 2011demonstrated the Batik method for making microfluidic channels and modified the process slightly by printing wax on paper and then drawing a pattern over it followed by transferring that wax to the fabric[18]. Computer software was used to design the pattern and then the print of the pattern was taken out for processing. In 2012, A Nilghaz et al. improved the wicking property of the cotton microfluidic channel by using Na2CO3 solution[3]. Peijing Wu in 2015 developed a colorimetric µCAD using a cotton cloth with hydrophobic channel barriers defined through the photolithography technique achieving channel resolution as small as 100 µm[19]. However, the method required costly clean room processing. Guan et al. in 2015 fabricated µCADs using wax screen printing method instead of wax-patterning technique and was much faster and required lower processing temperature [20]. In 2019, A Nilghaz et al. reported the use of wax printers (Xerox ColorQube 8570 printer) to print microfluidic patterns over fabric [21]. The fabric was attached to papers using the tapes and then it was inserted into the printer. Printed fabric was then heated in oven to melt the wax and ensure complete barrier formation throughout the cloth thicknesswherein channels widths of 200–500 µm obtained were blurred and the smallest nominal width of wax printed microfluidic channel reported was 1 mm on the fabric through this printing technique. Further, various low cost patterning methods like use of permanent markers, crayon wax, etc. over fabrics ashydrophobic barriers have also been explored unlike the above printing technique [22], [23]. Analytical devices developed using microfluidic cloth based technique is still at their initial stage although many researchers have demonstrated the development of various sensors over them [24, 25]. Benito-Lopez et al., in 2009 reported the first work on the development of a µCAD for monitoring sweat pH in real-time [26]. The device demonstrated real-time detection and monitoring of sweat pH during exercise using two µ-LEDs placed above and below the sensing area. Radha S. P. Malon et al in 2014 developed a fabric-based microfluidic structure for lactate measurement from saliva [27].The hydrophilic reaction zone on fabric was fabricated using wax-printing techniques and electrodes were developed using the template method. Guan et al. in 2016 from Zhang’s group were the first to demonstrate electrochemiluminescence detection in µCADs which were used for the detection of glucose levels in different samples [28].Very recently, Stojanovi et al. in the year 2020 reported a textile-based microfluidic system for the detection of cytostatic drug concentration CPA, in human sweat[15]. Cotton and polyester were used as fabric for components of the multi-layered microfluidic platform where higher concentrations of CPA in sweat resulted in a lower value of electrical resistance. Various cloth-based microfluidic functional components like microvalves, fluid velocity control elements, micromixers, and microfilters, etc have also been reviewed in detail by C. Zhang et al[14].

Beeswax, the oldest known thermoplastic material, exhibits a unique melting behavior that sets it apart from other homogeneous compounds. Instead of immediately melting upon heating, beeswax undergoes a series of intermediate states, transitioning from solid to plastic, semi-plastic, semi-liquid, and finally liquid. This gradual melting process commences around 40°C and is completed at approximately 64°C. This exceptional characteristic makes beeswax highly suitable for bonding applications requiring low temperatures. Notably, beeswax is a natural and renewable substance. However, due to variations in its chemical composition depending on factors such as bee species and geographical origin, beeswax proves to be an intriguing choice for manufacturing microfluidic devices. In a study by MaríaDíaz-Gonzalez et al., the fabrication of bio-functionalized microfluidic structures was achieved through the use of low-temperature wax bonding, highlighting the potential of beeswax in this field[29]. In a study conducted by Immanuel Nunut et al., the use of beeswax in printing technology for fabricating a paper-based microfluidic system was investigated[30]. The researchers extensively tested the fabricated channel by printing beeswax and verifying that it effectively penetrated the paper material, preventing the fluid from spreading out and ensuring controlled flow inside the channel. Their comprehensive testing confirmed the suitability of beeswax for creating functional microfluidic channels in paper-based devices.

Thus, the above literature reviews suggests that although substantial progress has been made in the field of capillary driven microfluidics over paper, successful realization of microchannels over fabrics are still in its native state. Moreover, upcoming wearable technology coupled with affordable healthcare demands the need for development of fabric based microfluidic diagnostic device through a low-cost, facile and easy-to-replicate technique for immediate realization and batch production. In this context, the current research aims to address this gap by optimizing fabrication steps to realize a cost-effective microfluidic device utilizing cloth or fabric materials using stencil patterns as channel masks and wax coating unlike the conventional printing technique. Optimization of a beeswax-based processing for realization of an efficient hydrophobic barrier over fabric material through the use of pre-designed PVC clear sheet has been discussed in the present study and compared with an insulation tape based masking method. Design and realization of complex microfluidic channels as well as micro-mixtures were successfully demonstrated. The technique was finally used to successfully realize fabric based devices for glucose detection applications through a rapid and simple, colorimetric testing method. This computed colorimetric data using image processing revealed the relation between glucose concentration and pixel intensity. The experimental results clearly indicate the potential of this approach as a low-cost diagnostic device compared to existing methods.

2.1 Materials and method

The µFADs were fabricated using a substrate material comprising 100% pure cotton fabric with a thread count of 110, sourced from Fabric Pandit-Surat, India. The channel barrier was constructed by repurposing PVC clear sheets obtained from TechNova (NovaStick Labels) and insulating tape as masks. This channel barrier was tested for both 100% organic pure unrefined beeswax from Hari Bee Farm in Tamil Nadu, India along with commercially available candle wax. The glucose oxidase (GO_x) enzyme, sourced from Aspergillus Niger, was procured from Sigma-Aldrich (St. Louis, MO, USA) for the fabrication of glucose sensor which exhibited an enzyme activity of 181 Umg^− 1. Horseradish peroxidase (HRP) with an enzyme activity of 73 Umg^− 1 and trehalosedihydrate was also purchased from Sigma-Aldrich. Chemicals such as glucose anhydrous, sodium phosphate monobasic anhydrous, and sodium phosphate dibasic anhydrous were obtained from Himedia Laboratories Pvt. Ltd. in Nashik, India. Potassium iodide (KI), with a molar mass of 166.01 gmol^− 1, was acquired from Nice Chemicals (P) Ltd, Kerala, India. All chemicals were used in their original forms, and deionized (DI) water of resistivity 18.2 MΩ was utilized in their preparation.

2.2 Fabrication optimization for development of µFADs

Development of microfluidic fabric based analytical devices (µFADs) was investigated in two parts. Initial studies were conducted using simple insulation tape based masking methods with candle wax as hydrophobic barriers. Inability of the process to develop efficient microchannels let to the exploration of a different masking technique using PVC clear sheets as well as use of beeswax as an alternative hydrophobic channel wall material due to its superior thermoplastic property enabling better seepage into the test substrate.

2.2.1 µFADs fabricated using insulation tape and candle wax

An 8 cm long microfluidic device was designed with a CAD tool and then printed on tracing paper. This was then put on top of a piece of carbon copy paper kept over an insulating tape. The image of the patterned microchannel on the tracing paper was drawn through a ruler by hand and was transferred over to the insulation tape surface through the carbon copy paper. The dimensions of the three sub-channels near the middle were each of3 mm width. Thereafter, the insulation tape was cut to size and stuck over a clean cloth surface followed by pressing it hard with a flat, smooth metal tool to ensure effective fixation of the tape over the cloth surface as shown in Fig. 1(a-i). The fabrication trials explored the consequences of application of single sided as well as double sided insulation tape. For the single sided insulation tape method, wax candle was lit, and the liquid wax was dropped onto the edges of the pattern formed using the tape as shown in Fig. 1(a-ii). This method does not require any complex heating procedure and was easy to perform. Subsequently, as indicated in Fig. 1(a-iii), excess wax was removed using a flat tool which also helped in pressing the wax deep inside the fabric. Finally, the tape was removed, and the channel was formed as shown in Fig. 1(a-iv).

Thereafter, opposite side of the microchannel was also explored to be masked with the insulation tape. Before the candle wax was put on top of it, a similar pattern was also cut out and stuck to the other side of the cloth surface. Liquid candle wax was melted and dropped onto the edges of the pattern first on one side and thereafter over the other side and pressed with a flat tool to ensure complete seepage of the molten wax across the cloth width. Figure 1(b) shows the complete microchannel obtained using the double-sided tape fixation technique. Figure 1(c) shows the effect of single and double-sided tape fixation on the dimensions of a linear channel of 2.5 cm length and 5 mm width.

Micro-mixing testing of the fabricated complex microchannels was performed with diluted orange and green color. Fluid samples were inserted through the two input terminals of the microchannel structure and the flow mixture pattern was recorded with test inks being added after every 10 seconds from the junction where inlet ports metuntil complete mixing as shown in Fig. 2.

Figure1. (a) Fabrication steps for realizing microfluidic channel with insulation tape and candle wax involving(i)fixation of channel pattern using insulation tape, (ii)wax coating over it, (iii)scrapping of excess wax and (iv)peeling-offof the pattern. (b) Effective channel structure with double-sided insulation tape. (c) Effect of single and double-sided insulation tape fixation over amicrochannel structure of dimension 0.5 cm x 2.5 cm. (d) Fabrication of complex microfluidic channel using PVC clear sheet and beeswax showing (i) fixation of PVC clear sheet over the fabric surface and (ii) fabric coated with beeswax to form a complex micromixer channel. (e) Optical microscopic image of fabricated microfluidic channel using insulation tape and candle wax-based showing the channel surface (i) before and (ii) after addition of diluted blue ink and the same fabricated using PVC clear sheet and beeswax showing the channel surface (iii) before and (iv) after addition of turmeric extract.

2.2.2µFADs fabricated using PVC clear sheet and beeswax.

Considering the drawback of the above insulation tape technique, involving crude method of hand-drawn microfluidic channel, a second method involving use of sticker-cutting tool over PVC clear sheets were employed as stencil masks to realize the microfluidic structures. To create this, the cotton fabric underwent a pre-treatment process involving immersion in hot water at 80°C for an hour to enhance capillary action of tests fluids[31]. Subsequently, the fabric was air-dried at room temperature for an additional hour. Using AutoCAD Fusion 360 software, the microfluidic pattern was designed with dimensions almost similar to those above and then printed onto a transparent PVC sheet using a sticker-cutting technique. This printed pattern was meticulously applied to both sides of the cotton cloth and thereafter pressed hard to ensure effective fixation of the PVC sheet as shown in Fig. 1(d-i). Following this, beeswax was melted at 75°C using a hotplate, and the fabric was coated with the molten beeswax. The coated fabric was then left to air-dry at room temperature for 15 minutes, resulting in complete drying of the beeswax followed by peeling-up of the PVC tape form the channel areas, as depicted in Fig. 1(d-ii).

Fabricated microchannels were also investigated for its suitability as µFADs. Initial testing involved droplet confinement of various test liquids over hydrophobic beeswax-based walls as shown in Fig. 3(a). These tests aimed to confirm the barrier's effectiveness with various test fluids and to ascertain the duration of fluid sample retention as well as type of fluids used when beeswax was employed as a hydrophobic barrier. A circular boundary measuring 2 cm in diameter on the cotton fabric was achieved by applying beeswax using the PVC clear sheet technique. Following this, the cotton fabric substrate was allowed to air dry and cool for ~ 5 minutes. Subsequently, single droplets of different organic test fluids like extracts of tomato, carrot, coffee, turmeric, mint leaf, and pomegranate were placed. Observations were made to detect any indications of leakage along the circular channel boundaries.

Microfluidic channels of different shapes (straight Y, zig-zag Y and complex) were successfully fabricated using the above technique and micromixing application was tested with them as shown in Fig. 3(b-d). To observe the mixing effect, two distinct test fluids(pomegranate and mint extract for straight Y channel, red and blue ink for the zig-zag Y channel and pomegranate and turmeric extract for the complex channel) were introduced into separate inlet reservoirs within the channel. Contrasting colors were opted for to enhance visibility. Upon completion of the flow, an additional drop of both colors was introduced at the same time through the two inlets promoting the convergence of fluids and the formation of a mixture. The resulting amalgam was examined, revealing the successful blending of different colors as the synthetic and natural extracts traversed the channels.

Further, experiments were also conducted to ascertain the minimum channel size that could be attained by this PVC stencil-beeswax technique by varying the channel dimensions from 4 mm to 500 µm as shown in Fig. 3(e). A blue ink was flown through the channel to indicate its efficacy after realization of the complete device and the results were compared for different microchannel widths.

2.3 Microscopic analysis:

The surface structure of the fabric was assessed both before and after undergoing pretreatment. Notably, the pretreatment substantially improved the material's wicking capabilities. Examining the application of candlewax and insulation tape under a microscope revealed inadequate wax penetration, leading to an uneven flow of diluted ink within the microfluidic channel. Conversely, using PVC clear sheet with beeswax resulted in a more uniform flow and proper wax penetration, consequently ensuring the channel's precise shape. As depicted in Fig. 1(f), the porous fabric structure was completely enveloped by beeswax, forming a thin, continuous layer.

2.4 Flow Testing of fabricated Microchannels

Imbibition of fluids through porous medium following capillary action is generally governed by the famous Lucas Washburn (L-W) model[32] which states that the distance travelled by the fluid inside a porous channel is proportional to the square root of the time it takes to flow through it and is given by Eq. (1) as:

l =$\sqrt{\left(\frac{{\gamma }\text{r}\text{c}\text{o}\text{s}\theta }{2\mu }\right)t}$(1)

where, l is the distance of the fluid front, γ the solution surface tension, r the effective pore radius of the paper, θ the solution contact-angle with the paper, µ the viscosity, and t the elapsed time.

In the present study, flow rate testing was performed for fluid flow over a straight microchannel to ascertain the applicability of L-W model. The Input ends were kept immersed inside an ink tank and gradual flow due to capillary action was observed as shown in Figure 4(a). The test sample was kept horizontal during the flow rate measurement process. The distance travelled by the fluids within the channel was recorded and plotted against time. Further, general flow rate testing over the various channels (complex) were tested for studying the difference (if any) of the flow rate through insulation tape technique and PVC sheet technique through periodic addition of colored inks (additional drops being added after every 40 seconds until complete mixing) unlike continuous immersion as in the prior case. The differences were noted as shown in Figure 4 and accounted for. Similar studies were also performed for straight Y and zig-zag Y shaped channel fabricated using PVC sheet for micro-mixing applications and the results obtained were noted and compared as shown in Figure 4.

2.5 Development of colorimetric-based glucose sensor using PVC clear sheet-beeswax:

PVC clear sheet based µFADs were further explored in development of a colorimetric based glucose assay due to their significant improvement in the obtained microchannel profiles. For the glucose assays, a combination of enzymes including glucose oxidase (GO_x) with an activity level of 120 Uml^− 1 and horseradish peroxidase (HRP) with an activity of 30 Uml^− 1 was prepared. This enzymatic mixture was formulated in a 100 mM PBS (pH = 6.0) solution, which acted as the environment for the enzymatic reactions essential for the assay. In order to carry out the assays, the enzymatic solution was accurately droppedto the detection zone and allowed to air-dry for 15 minutes.

Potassium iodide (KI) was used as the chromogenic agent with trehalose as a stabilizer for the same. 0.6M KI was mixed with0.3 M trehalose in 50 ml of DI water. The chromogenic agent was carefully applied to the designated zone using a micropipette and then allowed to dry for an additional 15 minutes. Following this, solutions containing varying amount of glucose concentration were introduced into the designated detection zone. The color intensity in the detection zone was observed to gradually vary with the glucose concentration variation.

The above obtained color intensity variation for the different glucose assays were captured and analyzed for quantitative estimation of glucose concentration through image processing. The images of different glucose assay were captured using smartphone camera (Google Pixel 6, 50 megapixel) under the same light conditions. After image acquisition, the images were pre-processed and glucose assay region was segmented out using Otsu thresholding [33]. Subsequently, the average intensity of the segmented region was calculated for each image in both gray scale and RGB. In gray scale analysis, the color image was converted to grayscale image and average pixel intensity of the segmented region was calculated. In RGB analysis, individual R, G and B component average values were computed. The image was saved as an 8-bit image, so the intensity values are in the range of 0 and 255. Afterwards, linear regression was performed and the equation of the straight line was computed to predict the relation between glucose concentration and pixel intensity.

3.1 Micromixer application of insulation tape & candle wax based µFADs:

Microchannels fabricated using insulation tape technique on both sides and candle wax were tested to demonstrate the micro-mixing process using a complex design as elucidated in the preceding section. This intricate pattern was then put to the test by introducing diluted ink of two distinct colors namely, orange, and green diluted ink into the input ports of the micro-mixer. As the diluted ink entered, it flowed across the channel walls due to capillary action of the porous structure of the cotton fabric.

Figure 2 (a-d) reveals an irregular flow pattern of orange and green diluted ink across various channels and sub-channels, where although both inks proceeded through the same distance as shown in Fig. 2(a), irregular division of sub-channels beyond Fig. 2(b) resulted in irregular distribution of flow pattern (Fig. 2(c)) with predominant orange ink in two sub-channels and green along only the third. Finally mixed output color was obtained near the opposite end as shown in Fig. 2(d) and (e). The above observation may be attributed to the fact that the fabricated channel had varying widths across its three arms (sub-channels) with dimensions varying between 3.5 mm to 1.5 mm for the actual 3 mm width channels as shown in Fig. 1(b).Thus, an increase as well as decrease in channel widths was both observed for the fabricated channels which might be possibly attributed to either incomplete penetration of wax through the fabric layer (for increased channel dimension) or over penetration under the tape mask (for reduced channel dimensions). These asymmetric channels designs, irregular pattern widths and improper flow of candle wax still posed severe limitation towards its practical application.

It was however evident through the above illustration that the application of a double-sided insulation tape applied on both sides of the fabric, resulted in a successful demonstration of a micromixer application. However, the inefficacy of the insulation tape in effectively preventing the penetration of wax under its desired zone resulted in formation of irregular channel walls and flow patterns, which might even result in channel blockage for very narrow ones. Consequently, the microfluidic channel's performance using double-sided insulation tape technique was compromised, representing a significant drawback of this method.

Figure 2. Flow testing of fluids within the complex micromixer channel with orange and green diluted ink following simultaneous addition of (a) 1st drop,(b) 2nd drop, (c)3rd drop and (d) 4th drop of both the inks.(Additional drops were added after every 10 seconds until complete mixing) (e) Demonstration of complete mixing of input green and orange color resulting in a brownish solution at the output ends of the complex channel..

3.2 Microfluidic application of PVC clear sheet & beeswax based µFADs:

In order to verify the efficacy of the fabricated PVC clear sheet-beeswax based µFADs, an initial assessment was carried out involving a droplet test method. Various test fluids like extracts of tomato, carrot, coffee, turmeric, mint leaf, and pomegranate were placed inside the circular channel barrier and observations were made for possible seepage of the test fluids outside the channel boundaries. All samples were observed to effectively confine the above natural extracts without any leakage even after 30 min from the time of their insertion. Figure 3(a) shows the fabrication steps involved in affixing the PVC clear sheet followed by coating of beeswax and final droplet confinement testing over them.
Fig. 3: (a) Droplet testing over fabric with circular wells showing (i) affixed circular shaped PVC clear sheet, (ii) fabric coated with beeswax to form a circular shape and (iii) testing with extracts of tomato, carrot, coffee, turmeric, mint leaf, and pomegranate juice. (b) Fabrication of straight Y shaped channel (4 cm length) and its testing using with pomegranate extract (red) and mint leaf (green) extract, (c) fabrication of zig-zag Y shaped channel (8 cm length)and its testing using red ink and blue ink and (d) fabrication of complex channel (8 cm length) and its testing using with pomegranate extract (red) and turmeric (yellow) extract. (e) Minimum channel width realization of ~ 500 µm using PVC stencils.

Subsequently, the PVC clear sheet based µFADs were fabricated and tested for micromixer application using a straight Y channel (4 cm in length), a zig-zag Y channel(8 cm in length) as well as the complex micromixer design (8 cm in length). From the observations in Fig. 3(b-d)it is clear that the samples demonstrated effective micromixing application between various test fluids (pomegranate and mint extract for straight Ychannel, red and blue ink for the zig-zag Y channel and pomegranate and turmeric extract for the complex channel) without the formation of any irregularities in channel shape and retaining almost uniform channel widths through the design structures. Further, beeswax also promoted easy and uniform coating of the wax as a hydrophobic boundary and was easily confined within the PVC clear sheet mask region. Thus, upon comparison of Fig. 1(b) and Fig. 3(d), it may be clearly observed that PVC clear sheet-beeswax techniques resulted in much better and efficient microfluidic channels of different complexities required for most µFADs.

Figure 3(e) demonstrates the minimum microchannel line width achievable using the above PVC-beeswax technique which was observed to be 500 µm over fabrics wherein the input blue ink was observed to flow smoothly with complete fluid confinement. Microscopic investigations also revealed proper channel walls with complete beeswax penetration into the fabric and no significant channel irregularities as shown in Fig. 1(e). This, to the best of the authors knowledge is the minimum achievable resolution over fabric so far and is comparable to that achieved using printing technique over fabrics as well [21].

Fig. 4. (a) Demonstration of L-W model based flow profile along a straight channel with input end dipped in red ink showing (b) non-linear dependency of distance travelled by the fluid with time. Inset shows the linear variation of flow with square root of time. Fluid flow along complex channel fabricated using (c) conventional insulation tape - candle wax and (d) PVC clear sheet – beeswax technique showing (e) variation of fluid flow over time as well as fluid flow along PVC clear sheet – beeswax based (f) straight Y and (g) zig-zag Y channel for gradual test fluids being added after specific time intervals.

3.3 Flow testing of microchannels

Experimental observations were also carried out in the present case to measure the flow rate as per the L-W technique. A straight microchannel was used to measure the flow rate of red ink as shown in Fig. 4(a) and the output data was plotted as shown in Fig. 4(b) which depicts the non-linear behavior of the flow pattern thereby validating the applicability of L-W model in the flow profile. Inset of Fig. 4(b) shows the linearized plot of distance traversed by the test fluid with square root of time.

The above results reveal that L-W model was applicable for fluid flow through fabrics which forms a basic guideline for design of µFADs. However, most microfluidics based diagnostic devices requires addition of few drops of the test fluid only instead of continuous test fluid sources. Thus, general flow testing through periodic addition of colored inks (additional drops being added after every 10 seconds until complete mixing) was also carried out unlike continuous immersion as in the prior case. Figure 4 (c-e) demonstrates the flow testing over the conventional candle wax-insulation tape channels (Fig. 4(c)) with those of beeswax-PVC sheet based channels (Fig. 4(d)). Here it may be noted that the flow time was recorded from the moment the different tests fluids met together at the 1st junction (for both Y channels and complex channel) until complete channel coverage with additional drops being added after every 10 seconds. This was done since there was an initial delay in wicking of test fluids at inlet ends and orientation of fabric threads (horizontal and perpendicular) delayed oblique flows along the arms of the inlet Y/complex channel. The average flow rate of the test fluids through the complex micromixer channels was plotted and measured to be ~ 0.43mm/sec and ~ 0.45mm/sec for candle wax-insulation tape type and PVC clear sheet-beeswax type respectively as observed through Fig. 4(e). Similarly, the average flow rate of the test inks through PVC clear sheet-beeswax based straight Y channel and zig-zag Y channel (Fig. 4(f) and (g)) was almost same and measured to be ~ 0.41mm/sec and ~ 0.42mm/sec respectively as shown in Fig. 4(h).

Further, although flow rates were almost similar for both complex channels in Fig. 4(c) and(d), flow patterns observed were highly non-homogenous across the insulation tape based complex channel with marked different colors in the three sub-channels indicating improper mixing of the test fluids even through chaotic advection. Interestingly, a homogenous mixture was noted only at the end of the complex channel where all three sub-channels met. On the other hand, complex channel over PVC clear sheet, demonstrated homogenous mixing right from the point where the three sub-channels started until the outlet end. This indicates effective demonstration of chaotic advection near the sub-channels through random transport of ink species by the laminar flow over the PVC based fabric microfluidic channels applicable in various passive micromixing techniques.

This effect of chaotic mixing was further noted for PVC based straight Y and zig-zag Y channels of Fig. 4(f) and (g). The zig-zag Y channel demonstrated micromixing of blue and red ink almost instantaneously and was homogeneously distributed till the end of the channel. However, for the straight Y channel, a gradual laminar flow was observed near the junction with complete diffusion of the blue and red ink almost towards the end of the channel. Thus, the above observations of flow tests over PVC sheet –beeswax based fabric microchannels are comparable to anyconventional polymer based microchannel performances which clearly demonstrate the suitability of exploring them as potential µFADs for development of upcoming low cost wearable products.

3.4 Colorimetric test of fabric-based glucose sensor and its Quantitative estimation

Effectiveness of the PVC clear sheet –beeswax based microchannels was further explored to develop colorimetric based µFAD for glucose sensing application. The process of fabricating the µFAD is illustrated schematically in Fig. 5(a). The detection zone displays discernible shifts in color that correspond to varying glucose concentrations. When glucose is introduced into the reaction zone, the catalytic function of Glucose Oxidase (GO_x) converts glucose and oxygen into gluconic acid and hydrogen peroxide (H₂O₂). Subsequently, with the assistance of Horseradish Peroxidase (HRP), the H₂O₂ oxidizes Iodide (I-) to yield a purple iodine indicator and a dark brown trehalose indicator [31, 32]. The color intensity in the detection zone gradually transitions as the glucose concentration changes. Specifically, a dark brown color with hints of purple denotes 50 mM glucose concentration while a brownish grey shade corresponds to 40 mM concentration. A purple color signifies a glucose concentration of 30mM, while a light brown hue corresponds to 20mM glucose, and finally, a light purple hue indicates a glucose concentration of 10mM as depicted in Fig. 5(b). This colorimetric response furnishes a firsthand visual method for quantifying glucose levels and facilitates the identification of varying glucose concentrations within the tested samples.

Fig. 5. (a) Fabrication process steps of circular test patches for realization of colorimetric glucose sensor showing (i) untreated piece of cotton fabric, (ii) treated fabric with hot water, (iii) patterning of PVC clear sheet using sticker cutting method and pasting of the same over fabric, (iv) coating of beeswax in the concentric ring creating a circular hydrophobic wall, (v) peeling off of PVC pattern following coating of beeswax, (vi) spotting of both enzymatic and colorimetric reagents in the circular area on the surface of the fabric, (vii) addition of glucose analyte sample into the testing spot. (b) Sequence of color changes observed for varying glucose concentration from 50mM to 10 mM over the fabricated fabric based circular test patch and (c) Plot of intensity variation of colorimetric data against different levels of glucose concentration for (i) Red color intensity (ii) Green color intensity (iii) Blue color intensity (iv) Black & White color intensity.

The quantitative estimation of the glucose concentration was performed by segmenting out the glucose assay region using Otsu thresholding in all the images. Consequently, the average intensity of the segmented region was computed in both gray scale and RGB. The images were saved as 8-bit image i.e., 0 stands for black and 255 stands for white. However, for ease of interpretation, the measured pixel intensity was subtracted from 255 for all the cases. Subsequently, the average intensity values were plotted against the glucose concentration. It is evident from the plot that average intensity and glucose concentration has a linear dependency. Therefore, linear regression has been done and the least square straight line is computed for gray scale and R, G and B component values. Figure 5(c) illustrates the experimental results of the intensity as a function of the glucose concentration (N = 4). The dashed lines represent linear curve fit to the intensity-concentration data. The regression equations are given below.

$${I}_{r}=2.4693m+101.2630 ({R}^{2}=0.94899)$$

$${I}_{g}=2.3034m+115.4225 ({R}^{2}=0.94515)$$

$${I}_{b}=2.6705m+105.0707 ({R}^{2}=0.95288)$$

$${I}_{bw}=2.3283m+112.7596 ({R}^{2}=0.93912)$$

where, I_r, I_g, I_b are the calculated intensity of R, G and B component of the color image; I_bw is the calculated intensity of the corresponding grayscale image and m is the glucose concentration. High R² value indicates good fit of the regression lines. These experimental results show potential of the proposed method towards low-cost diagnostic device development for glucose measurements.

The realm of fabric-based devices presents several benefits, including flexibility, affordability, and the advantages of microfluidic platform for real-time testing of bodily fluids like blood, sweat, saliva, mucus, and urine in reduced quantities for any analyte detection. Demand for a rapid low-cost fabrication technique still pose significant challenge towards its practical usage for seamless integration with other wearable devices particularly for fabric based microfluidic devices. Present article highlights the use of PVC clear sheet as a stencil as an alternative strategy to develop microfluidic devices. In addition, beeswax was used as the channel barrier unlike convention candle wax due to its superior thermoplastic property enabling better seepage into the test substrate for channel wall formation.Conventional wax based printing tools or wax based screen printing technique requires the wax (ink) to be maintained at a particular temperature range to allow for smooth flow for effective printability. On the other hand, present system involves easy realization of the channel masks using laser based sticker-cutting method over PVC clear sheets followed by sticking over the fabric surface, wax coating and scrapping the excess. This rapid and simple technique for realization of channel masks with beeswax as barriers resulted in facile realization of various complex microchannels, microwells and micromixers, eliminating the need for a controlled wax temperature. The minimum channel width that was successfully achieved with this technique was 500 µm over fabric which has not been reported by any other group so far and is comparable to that of the printing technique as well. Further, these PVC clear sheet stickers were compared with conventional tape based technique to act as adhesive masks for microfluidic channels and superiority of the former was accounted for.

Fabricated microwells demonstrated successful confinement of these microwells towards various organic and inorganic fluids demonstrating its potential application in various chemical and pharmaceutical fields. Microchannel test results showed effective demonstration of various flow properties like effective confinement of test fluids, successful flow testing through various microchannel profiles obeying L-W flow model as well as complex micromixing operations with faster micromixing over zig-zag Y channels representing chaotic advection (compared to straight Y channels)ascertaining its applicability as a potential µFADs. Finally, the study focused on the development of a fabric-based colorimetric biosensor tailored for glucose monitoring in point-of-care scenarios. These fabric-based biosensors exhibited fair sensitivity and detectability, facilitating the analysis of glucose concentrations over a broad range (10 mM to 50 mM) through colorimetric assay. Image processing of these captured colorimetric data through smart phone successfully provided a quantitative estimation of the glucose content through a good linear curve fit with an R² value of >0.93. This in turn can be used for successful interpretation of any colorimetric intensity value to the unknown glucose concentration thereby justifying utility of the present fabric based micro-assay as a potential µFAD. Overall, this study underscores the promising potential of the new sticker cutting technique over PVC clear sheets with beeswax as practically realizable fabric-based colorimetric biosensors for point-of-care wearable applications.

Author contributions

LP: conceptualization, methodology, investigation of PVC clear sheet-beeswax based µFAD and writing of original draft. AS, PG, SK and VJ: methodology and investigation of insulation tape-candle wax based method and writing of initial manuscript draft. RP: investigation and interpretation of colorimetric data. LP: writing – review & editing of the manuscript. DM: supervision, writing – review & editing of the manuscript.

Conflicts of interest

There are no conflicts to declare.

A. C. R. Grayson et al., “A BioMEMS review: MEMS technology for physiologically integrated devices,” Proc. IEEE, vol. 92, no. 1, pp. 6–21, 2004.
A. K. Au, H. Lai, B. R. Utela, and A. Folch, “Microvalves and micropumps for BioMEMS,” Micromachines, vol. 2, no. 2, pp. 179–220, 2011.
A. Nilghaz, D. H. Wicaksono, D. Gustiono, F. A. A. Majid, E. Supriyanto, and M. R. A. Kadir, “Flexible microfluidic cloth-based analytical devices using a low-cost wax patterning technique,” Lab. Chip, vol. 12, no. 1, pp. 209–218, 2012.
P. Gravesen, J. Branebjerg, and O. S. Jensen, “Microfluidics-a review,” J. Micromechanics Microengineering, vol. 3, no. 4, p. 168, 1993.
J. Hwang, Y. H. Cho, M. S. Park, and B. H. Kim, “Microchannel Fabrication on Glass Materials for Microfluidic Devices,” Int. J. Precis. Eng. Manuf., vol. 20, no. 3, pp. 479–495, Mar. 2019, doi: 10.1007/s12541-019-00103-2.
D. C. Duffy, J. C. McDonald, O. J. A. Schueller, and G. M. Whitesides, “Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane),” Anal. Chem., vol. 70, no. 23, pp. 4974–4984, Dec. 1998, doi: 10.1021/ac980656z.
J. C. McDonald and G. M. Whitesides, “Poly(dimethylsiloxane) as a Material for Fabricating Microfluidic Devices,” Acc. Chem. Res., vol. 35, no. 7, pp. 491–499, Jul. 2002, doi: 10.1021/ar010110q.
S. K. Sia and G. M. Whitesides, “Microfluidic devices fabricated in Poly(dimethylsiloxane) for biological studies,” ELECTROPHORESIS, vol. 24, no. 21, pp. 3563–3576, Nov. 2003, doi: 10.1002/elps.200305584.
M. Talebi, K. Cobry, A. Sengupta, and P. Woias, “Transparent glass/su8-based microfluidic device with on-channel electrical sensors,” in Proceedings, MDPI, 2017, p. 336. Accessed: Dec. 20, 2023. [Online]. Available: https://www.mdpi.com/2504-3900/1/4/336
S. Kanishka, P. Srichandan, A. Tiwari, A. Shukla, and D. Maji, “Fabrication of 2D/3D Paper based Microfluidic Channels using Canvas Sheets and Tissue Papers,” in 2021 IEEE Mysore Sub Section International Conference (MysuruCon), IEEE, 2021, pp. 477–481. Accessed: Dec. 20, 2023. [Online]. Available: https://ieeexplore.ieee.org/abstract/document/9641602/
A. W. Martinez, S. T. Phillips, G. M. Whitesides, and E. Carrilho, “Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices,” Anal. Chem., vol. 82, no. 1, pp. 3–10, Jan. 2010, doi: 10.1021/ac9013989.
A. T. Singh, D. Lantigua, A. Meka, S. Taing, M. Pandher, and G. Camci-Unal, “based sensors: Emerging themes and applications,” Sensors, vol. 18, no. 9, p. 2838, 2018.
V. Soum, S. Park, A. I. Brilian, O.-S. Kwon, and K. Shin, “Programmable paper-based microfluidic devices for biomarker detections,” Micromachines, vol. 10, no. 8, p. 516, 2019.
C. Zhang, Y. Su, Y. Liang, and W. Lai, “Microfluidic cloth-based analytical devices: Emerging technologies and applications,” Biosens. Bioelectron., vol. 168, p. 112391, 2020.
G. M. Stojanović et al., “A textile-based microfluidic platform for the detection of cytostatic drug concentration in sweat samples,” Appl. Sci., vol. 10, no. 12, p. 4392, 2020.
P. Bhandari, T. Narahari, and D. Dendukuri, “‘Fab-Chips’: a versatile, fabric-based platform for low-cost, rapid and multiplexed diagnostics,” Lab. Chip, vol. 11, no. 15, pp. 2493–2499, 2011.
F. Vatansever et al., “Toward Fabric-Based Flexible Microfluidic Devices: Pointed Surface Modification for pH Sensitive Liquid Transport,” ACS Appl. Mater. Interfaces, vol. 4, no. 9, pp. 4541–4548, Sep. 2012, doi: 10.1021/am3008664.
A. Nilghaz, D. H. Wicaksono, and F. A. A. Majid, “Batik-inspired wax patterning for cloth-based microfluidic device,” in 2011 2nd International Conference on Instrumentation Control and Automation, IEEE, 2011, pp. 82–86. Accessed: Dec. 20, 2023. [Online]. Available: https://ieeexplore.ieee.org/abstract/document/6130134/
P. Wu and C. Zhang, “Low-cost, high-throughput fabrication of cloth-based microfluidic devices using a photolithographical patterning technique,” Lab. Chip, vol. 15, no. 6, pp. 1598–1608, 2015.
W. Guan, C. Zhang, F. Liu, and M. Liu, “Chemiluminescence detection for microfluidic cloth-based analytical devices (μCADs),” Biosens. Bioelectron., vol. 72, pp. 114–120, 2015.
A. Nilghaz, X. Liu, L. Ma, Q. Huang, and X. Lu, “Development of fabric-based microfluidic devices by wax printing,” Cellulose, vol. 26, no. 5, pp. 3589–3599, Mar. 2019, doi: 10.1007/s10570-019-02317-z.
A. Chakrabarti, N. Narayan, A. J. Jacob, and D. Maji, “Filter Paper based Micromixer using Wax Crayon as Channel Barriers,” in 2021 IEEE 9th Region 10 Humanitarian Technology Conference (R10-HTC), IEEE, 2021, pp. 01–06. Accessed: Dec. 20, 2023. [Online]. Available: https://ieeexplore.ieee.org/abstract/document/9641690/
N. K. Mani, A. Prabhu, S. K. Biswas, and S. Chakraborty, “Fabricating paper based devices using correction pens,” Sci. Rep., vol. 9, no. 1, p. 1752, 2019.
M. Chuang et al., “Textile‐based Electrochemical Sensing: Effect of Fabric Substrate and Detection of Nitroaromatic Explosives,” Electroanalysis, vol. 22, no. 21, pp. 2511–2518, Nov. 2010, doi: 10.1002/elan.201000434.
Y.-L. Yang, M.-C. Chuang, S.-L. Lou, and J. Wang, “Thick-film textile-based amperometric sensors and biosensors,” Analyst, vol. 135, no. 6, pp. 1230–1234, 2010.
F. Benito-Lopez, S. Coyle, R. Byrne, A. Smeaton, N. E. O’Connor, and D. Diamond, “Pump less wearable microfluidic device for real time pH sweat monitoring,” Procedia Chem., vol. 1, no. 1, pp. 1103–1106, 2009.
R. S. Malon, K. Y. Chua, D. H. Wicaksono, and E. P. Córcoles, “Cotton fabric-based electrochemical device for lactate measurement in saliva,” Analyst, vol. 139, no. 12, pp. 3009–3016, 2014.
W. Guan, M. Liu, and C. Zhang, “Electrochemiluminescence detection in microfluidic cloth-based analytical devices,” Biosens. Bioelectron., vol. 75, pp. 247–253, 2016.
M. Díaz-González and A. Baldi, “Fabrication of Biofunctionalized Microfluidic Structures by Low-Temperature Wax Bonding,” Anal. Chem., vol. 84, no. 18, pp. 7838–7844, Sep. 2012, doi: 10.1021/ac301512f.
I. Nunut, Y. Whulanza, and S. Kassegne, “Testing of Beeswax Printing Technology in the Design of a Paper-Based Microfluidic System,” Int. J. Technol., vol. 11, no. 5, pp. 1036–104, 2020.
G. Zhu, J. Militky, Y. Wang, B. V. Sundarlal, and D. Křemenáková, “Study on the wicking property of cotton fabric,” Fibres Text. East. Eur., no. 2 (110), pp. 137–140, 2015.
D. dos S. de Abreu, M. Strauss, and M. Santhiago, “Flexible cellulose-based devices for monitoring physical parameters,” in Comprehensive Analytical Chemistry, vol. 89, Elsevier, 2020, pp. 361–395. Accessed: Dec. 20, 2023. [Online]. Available: https://www.sciencedirect.com/science/article/pii/S0166526X20300027.
N. Otsu, “A threshold selection method from gray-level histograms,” IEEE Trans. Syst. Man Cybern., vol. 9, no. 1, pp. 62–

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Realization of Beeswax-based Fabric Microfluidic Channels using PVC Clear Sheet Stencils

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Abstract

Figures

1. Introduction

2. Experimental Work

2.1 Materials and method

2.2 Fabrication optimization for development of µFADs

2.2.1 µFADs fabricated using insulation tape and candle wax

2.2.2µFADs fabricated using PVC clear sheet and beeswax.

2.3 Microscopic analysis:

2.4 Flow Testing of fabricated Microchannels

2.5 Development of colorimetric-based glucose sensor using PVC clear sheet-beeswax:

3. Results and Discussion

3.1 Micromixer application of insulation tape & candle wax based µFADs:

3.2 Microfluidic application of PVC clear sheet & beeswax based µFADs:

3.3 Flow testing of microchannels

3.4 Colorimetric test of fabric-based glucose sensor and its Quantitative estimation

4. Conclusions

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References

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