The authors acknowledge the financial support from the programs of the Natural Science Foundation of the Jiangsu Higher Education (22KJB460033), and Jiangsu Science and Technology Programme - Young Scholar (BK20200251). This work is also partially supported by the XJTLU AI University Research Centre, Jiangsu Province Engineering Research Centre of Data Science and Cognitive Computation at XJTLU and SIP AI innovation platform (YZCXPT2022103). The support from State Key Laboratory for Manufacturing Systems Engineering via the open project (SKLMS2023019) and Key Laboratory of Bionic Engineering, Ministry of Education, are also acknowledged.

1. Microembossing process for microchannel patterning on nanopaper
<ol>
	<li>Mold preparation 
	NOTE: Refer to Yuan et al.12 for details on mold preparation.
	<ol>
		<li>Prepare a PTFE film as indicated in the Table of Materials.</li>
		<li>Laser-cut the prepared PTFE film to make a convex microchannel mold (Figure 1A-I). 
		NOTE: The dimensions of the PTFE mold determine the microchannel dimensions (Figure 2E,F) in a linear first-order function relationship.</li>
	</ol>
	</li>
	<li>Nanopaper preparation
	<ol>
		<li>Disperse 4.0 g of (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-oxidized NFC gel (see Table of Materials) in distilled water (final concentration of 0.1 wt%).</li>
		<li>Stir the suspension heavily at 120.8 x g for 30 min at room temperature until no cellulose floc is visible.</li>
		<li>Vacuum-filter the clear suspension to obtain a nanopaper gel (Figure 1A-II). 
		NOTE: In this example, the diameter of the obtained nanopaper gel is 4 cm. NanoPADs can be tailored for various applications by selecting suction filtration devices with different radii, enabling the design of NanoPADs at different scales.</li>
	</ol>
	</li>
	<li>Embossing of nanopaper gel
	<ol>
		<li>Place the PTFE mold on the surface of the nanopaper gel.</li>
		<li>Emboss the nanopaper gel (Figure 1A-III) using the PTFE mold by the hot press for 10 min each time under optimized pressure and temperature (Figure 2A-D). 
		NOTE: Higher embossing pressure (250 kPa to 1000 kPa) improves fabrication accuracy but should not exceed 1000 kPa to prevent damage to the cellulose structure. Higher embossing temperatures (25-100 &#176;C) enhance microchannel accuracy by promoting dehydration and decarburization, but temperatures should not exceed 75 &#176;C to avoid gel wrinkling and reduced light transmittance7. In this example, optimized embossing parameters were 750 kPa and 75 &#176;C.</li>
	</ol>
	</li>
	<li>Mold releasing
	<ol>
		<li>Peel off an additional layer of filter nanopaper gel from the filter membrane (Figure 1A-IV).</li>
	</ol>
	</li>
	<li>Bonding
	<ol>
		<li>Attach the peeled layer on top of the embossed layer of nanopaper gel, stacking the two layers to create a hollow microchannel structure (Figure 1A-V). 
		NOTE: The stronger hydrogen bonding in &#39;gel-like&#39; nanopaper compared to fiber suspension and dried nanopaper enhances the entanglement and adhesion of nanocellulose fibers. Consequently, two layers of &#39;gel-like&#39; nanopaper can bond tightly through self-diffusion without external force.</li>
	</ol>
	</li>
	<li>Drying
	<ol>
		<li>Place the two layers of nanopaper gel in a drying oven at 75 &#176;C for approximately 30 min (Figure 1A-VI).</li>
	</ol>
	</li>
</ol>
2. Construction of fundamental microfluidic devices
<ol>
	<li>Construction of laminar-mixer
	<ol>
		<li>Prepare the NanoPADs with straight and curved channels (Figure 3A) following step 1. 
		NOTE: In this example, the dimensions of channels are 1 mm width and 50 &#181;m depth.</li>
		<li>Add red and blue droplets in the inlet zones simultaneously, allowing flow through the hollow channel automatically. 
		NOTE: The successful independent flow of the red and blue solutions in a straight channel and their mixing at the end of the curved channel can be attributed to the low Reynolds number of the layers in microfluidic devices and the radial flow induced by shear stress35.</li>
	</ol>
	</li>
	<li>Construction of droplet generator
	<ol>
		<li>Prepare the two-inlet NanoPADs with a T-junction channel (Figure 3D) according to step 1.</li>
		<li>Introduce water and hexadecane (oil), two immiscible liquids, into the two-inlet zones of the T-junction channel to generate droplets (Figure 3E). 
		NOTE: In this example, the dimensions of the T-junction channel are 1 mm width, 25 mm length, and 50 &#181;m depth.</li>
		<li>Fix the speed of Q1 at 6 &#181;L/min, and the speed of Q2 at n &#215; Q1 (n = 1-6). Use two syringe pumps and set them at the above speed to inject water and oil. This behavior is governed by the one simple scaling equation (provided below). 
		NOTE: In this example, oil and colored water were poured into the channel36. 
		<img alt="Equation 1" src="/files/ftp_upload/65965/65965eq01.jpg" style="margin: auto;" /> 
		Where &#945; = 1, &#946; = 1, L is the length, W is the width of the droplet, and Q1 and Q2 are the flow rates of water and hexadecane, respectively37,38.</li>
	</ol>
	</li>
</ol>
3. In-situ AgNP growth
<ol>
	<li>NanoPADs preparation
	<ol>
		<li>Prepare two-inlet NanoPADs with a converging detection zone (Figure 4A) according to step 1.</li>
	</ol>
	</li>
	<li>Successive ionic layer adsorption and reaction process
	<ol>
		<li>Prepare a 20 mM AgNO3 solution and a 20 mM NaBH4 solution (see Table of Materials).</li>
		<li>Drop 5 &#181;L of the 20 mM AgNO3 solution into the left inlet zone of the flow channel.</li>
		<li>Allow the AgNO3 solution to remain in the reaction zone for 30 s. 
		NOTE: Repeat steps 3.2.2. and 3.2.3. five times to ensure the uniform distribution of AgNPs without agglomeration, which could explain the higher band intensity.</li>
		<li>Drop 5 &#181;L of distilled water into the left inlet zone of the flow channel for rinsing. 
		NOTE: Repeat step 3.2.4. three times to ensure the removal of excessive, unadsorbed Ag ions through washing.</li>
		<li>Add 5 &#181;L of the 20 mM NaBH4 solution to the right inlet zone of the flow channel. 
		NOTE: Repeat step 3.2.5. until the AgNPs are generated evenly in the reaction zone. The chemical reactions involved in step 3 are represented by the following formula39: 
		<img alt="Equation 2" src="/files/ftp_upload/65965/65965eq02.jpg" style="margin: auto;" /> 
		 
		In this example, dense, uniform, well-structured AgNP arrays were formed on the NanoPADs (Figure 4B). The average diameter of the AgNPs was 55 nm (Figure 4C).</li>
	</ol>
	</li>
</ol>
4. SERS measurement
<ol>
	<li>Raman spectroscopy system preparation
	<ol>
		<li>Turn on the laser and launch the accompanying software for the Raman spectrometer (see Table of Materials).</li>
		<li>Employ a 50x objective for focusing and collecting Raman signals and a 532 nm laser for excitation.</li>
		<li>Set the spectral resolution to 2 cm-1 for accurate measurement. Set Raman spectrum measurement range from 400 cm-1 to 600 cm&#8722;1.</li>
		<li>Calibrate the Raman spectrometer using a silicon wafer12. 
		NOTE: Perform step 4.1. for step 4.2.</li>
	</ol>
	</li>
	<li>Rhodamine B (RhB) measurement
	<ol>
		<li>Dissolve 4.7 mg of RhB (see Table of Materials) in 10 mL of ethanol to prepare a 1 mM RhB solution.</li>
		<li>Prepare a series of RhB solutions with concentrations ranging from 10 &#181;M to 0.1 pM by diluting the 1 mM RhB solution in the ethanol.</li>
		<li>Add 5 &#181;L of the RhB solution onto the inlet zone of the NanoPADs channel and let it dry. 
		NOTE: Repeat step 4.2.3. for RhB solutions of different concentrations indicated in step 4.2.2.</li>
		<li>Set the excitation time to 10 s, the grating to 2 cm&#8722;1, and the number of cycles to 1. Set Raman spectrum measurement range from 500 cm-1 to 1800 cm&#8722;1.</li>
		<li>Adjust the coarse focus screw and fine focus screw individually to achieve proper focus, then click on stop to save the position.</li>
		<li>Click on start to begin the measurement.</li>
		<li>Repeat the measurements seven times and save the collected data.</li>
		<li>Turn off the laser.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Import the saved data into the data analysis software (see Table of Materials).</li>
		<li>Calculate the average spectrum from the saved data.</li>
		<li>Select the line draft option to plot Raman spectra.</li>
		<li>Utilize the Peak analyzer tool to set the baseline of the spectra.</li>
		<li>Apply the Signal process - Smooth function to smooth the spectra for final results.</li>
	</ol>
	</li>
</ol>

Micro-Embossed Fabrication of Nanocellulose Paper-Based Analytical Devices  

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T. H., Dien, N. D., Vu, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+synthesis+of+silver/gold+alloy+nanoparticles+for+ultra-sensitive+rhodamine+B+detection.">Facile synthesis of silver/gold alloy nanoparticles for ultra-sensitive rhodamine B detection.</a> RSC Advances. 11 (35), 21475-21488 (2021).</li><li>Li, D., Li, D. W., Li, Y., Fossey, J. S., Long, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+electroplating+and+stripping+of+silver+on+Au@SiO2+core/shell+nanoparticles+for+sensitive+and+recyclable+substrate+of+surface-enhanced+Raman+scattering.">Cyclic electroplating and stripping of silver on Au@SiO2 core/shell nanoparticles for sensitive and recyclable substrate of surface-enhanced Raman scattering.</a> Journal of Materials Chemistry. 20 (18), 3688-3693 (2010).</li><li>Sun, C. H., Wang, M. L., Feng, Q., Liu, W., Xu, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering+(SERS)+study+on+Rhodamine+B+adsorbed+on+different+substrates.">Surface-enhanced Raman scattering (SERS) study on Rhodamine B adsorbed on different substrates.</a> Russian Journal of Physical Chemistry A. 89 (2), 291-296 (2015).</li></ol>

The authors have nothing to disclose.

The primary focus of this study is to develop a simple method for fabricating microchannels on nanopaper. An efficient embossing technique was devised using PTFE as the mold to address this challenge12. By optimizing the temperature and embossing pressure, a series of experiments were conducted to establish a reliable fabrication process for NanoPADs. Additionally, the use of a quick-reference table to adjust the applications of NanoPADs in different fields was demonstrated. Although this method i...

Recently, nanofibrillated cellulose (NFC) paper (nanopaper) has emerged as a highly promising substrate material for various applications such as flexible electronics, energy devices, and biomedicals1,2,3,4. Derived from natural plants, nanopaper is cost-effective, biocompatible, and biodegradable, making it an appealing alternative to traditional cellulose paper5,6. Its exceptional properties include an ultra-smooth surface with a surface roughness of less than 25 nm and a dense cellulose matrix structure, allowing for the creation of highly structured nanostructures7. Abundant hydroxyl groups of nanopaper contribute to its compact and tightly packed nanocellulose structure8. Nanopaper exhibits excellent optical transparency and minimal optical haze, making it well-suited for optical sensors. Additionally, its inherent hydrophilicity enables pump-free flow, even with its thick structure, providing autonomous fluid motion9,10. Nanocellulose has diverse applications in biological sensors, conductive electronic devices, cell culture platforms, supercapacitors, batteries, and more, showcasing its versatility and potential11,12. Particularly, nanocellulose is promising for paper-based analytical microfluidic devices (&#181;PADs), offering unique advantages over conventional chromatography paper.
In the past decade, &#181;PADs have achieved significant attention due to their affordability, biocompatibility, pump-free operation, and ease of production13,14. These devices have emerged as effective point-of-care diagnostic tools, particularly in resource-limited settings15,16,17. A significant advancement in this field was the development of wax printing, pioneered by George Whitesides18 and the Bingcheng Lin group19, enabling the creation of functional &#181;PADs by incorporating microchannels on chromatography paper. Subsequently, &#181;PADs rapidly evolved, and various biosensing techniques, including electrochemical methods20, chemiluminescence21, and enzyme-linked immunosorbent assay (ELISA)22,23,24, were successfully implemented for the detection of diverse biomarkers such as proteins25,26, DNAs27,28, RNAs29,30, and exosomes31. Despite these achievements, &#181;PADs still face challenges, including slow flow speeds and solvent evaporation.
Several methods have been proposed for creating microchannels on nanopaper32,33,34. One approach involves 3D printing sacrificial ingredients into the material, but it requires a hydrophobic coating that limits pump-free operation33. Another technique involves manually stacking channel layers between nanopaper sheets using glue, which is labor-intensive32. Alternatively, spray-coating nanocellulose fibers onto pre-patterned molds can create microchannels, but it involves time-consuming and expensive mold preparation34. Notably, these methods are limited to millimeter-scale microchannels, compromising the advantages of microfluidic devices regarding reagent volume consumption and integration. Developing a simple nanopaper microchannel patterning process with micrometer-scale resolution remains a challenge.
This study presents a unique nanopaper microchannel patterning method based on practical microembossing. The approach offers several advantages over existing methods, as it requires no expensive or specialized equipment, is simple, cost-effective, and highly accurate. A convex microchannel mold is fabricated by laser cutting a polytetrafluoroethylene (PTFE) film, known for its chemical inertness and nonstick properties. This mold is then used to emboss microchannels onto a nanopaper gel membrane. A second layer of nanopaper gel is applied on top to create closed hollow channels. Using this patterning technique, fundamental microfluidic devices on nanopaper are developed, including a laminar mixer and droplet generator. Additionally, the fabrication of surface-enhanced Raman microscopy (SERS) NanoPADs is demonstrated. In-situ creation of a silver nanoparticle-based SERS substrate is achieved by introducing two chemical reagents (AgNO3 and NaBH4) into the channels, resulting in a remarkable performance with low limits of detection (LODs).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AgNO3&#160;</td>
			<td>Hushi (Shanghai, China)</td>
			<td>7761-88-8</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Hushi (Shanghai, China)</td>
			<td>64-17-5</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Hexadecane</td>
			<td>Macklin (Shanghai, China)</td>
			<td>544-76-3</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>LabSpec software</td>
			<td>Horiba (Japan)</td>
			<td>LabSpec5</td>
			<td></td>
		</tr>
		<tr>
			<td>Melamine</td>
			<td>Macklin (Shanghai, China)</td>
			<td>108-78-1</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Aladdin (Shanghai, China)</td>
			<td>16940-66-2</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Origin lab software</td>
			<td>OriginLab (USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET)&#160;</td>
			<td>Myers Industries (Akron, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene films</td>
			<td>Shenzhen Huashenglong plastic material Co., Ltd. (Shenzhen, China)</td>
			<td></td>
			<td>Teflon film</td>
		</tr>
		<tr>
			<td>PVDF filter membrane</td>
			<td>EMD Millipore Corporation (USA)</td>
			<td>VVLP04700</td>
			<td>pore size: 0.1 &#956;m</td>
		</tr>
		<tr>
			<td>Raman spectrometer</td>
			<td>Horiba (Japan)</td>
			<td>Xplo RA</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B</td>
			<td>Macklin (Shanghai, China)</td>
			<td>81-88-9</td>
			<td>&#62;95%</td>
		</tr>
		<tr>
			<td>Scanning electron microscopy (SEM)</td>
			<td>FEI(USA)</td>
			<td>Scios 2 HiVac</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Horiba (Japan)</td>
			<td></td>
			<td>diameter: 5 mm</td>
		</tr>
		<tr>
			<td>TEMPO-oxidized NFC slurry</td>
			<td>Tianjin University of Science and Technology</td>
			<td></td>
			<td>1.0 wt% solid, carboxylate level 2.0 mmol/g solid, average nanofiber diameter: 10 nm</td>
		</tr>
	</tbody>
</table>

microembossing: a convenient process for fabricating microchannels on nanocellulose paper-based microfluidics

Nanopaper, derived from nanofibrillated cellulose, has generated considerable interest as a promising material for microfluidic applications. Its appeal lies in a range of excellent&#160;qualities, including an exceptionally smooth surface, outstanding optical transparency, a uniform nanofiber matrix with nanoscale porosity, and customizable chemical properties. Despite the rapid growth of nanopaper-based microfluidics, the current techniques used to create microchannels on nanopaper, such as 3D printing, spray coating, or manual cutting and assembly, which are crucial for practical applications, still possess certain limitations, notably susceptibility to contamination. Furthermore, these methods are restricted to the production of millimeter-sized channels. This study introduces a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanopaper, achieving a minimum width of 200 &#181;m. The developed microchannel outperforms existing approaches, achieving a fourfold improvement, and can be fabricated within 45 min. Furthermore, fabrication parameters have been optimized, and a convenient quick-reference table is provided for application developers. The proof-of-concept for a laminar mixer, droplet generator, and functional nanopaper-based analytical devices (NanoPADs) designed for Rhodamine B sensing using surface-enhanced Raman spectroscopy was demonstrated. Notably, the NanoPADs exhibited exceptional performance with improved limits of detection. These outstanding results can be attributed to the superior optical properties of nanopaper and the recently developed accurate microembossing method, enabling the integration and fine-tuning of the NanoPADs.

Author Spotlight: Revolutionizing Microfluidics Through Microchannel Fabrication on Nanopaper 

Microembossing: A Convenient Process for Fabricating Microchannels on Nanocellulose Paper-Based Microfluidics

Our research introduces nanopaper as an innovative substrate, in contrast to conventional paper. Nanopaper offers optical transparency, exceptional smoothness, and chemical adaptability. Now the substrate currently lacks an efficient microchannel fabrication method, limiting its broader adoption in microfluidics.The progression of manufacturing technologies, including laser cutting and micro-or nano-3D printing has significantly improved motor precision. Consequently, it has simplified and enhanced the precision of microfluidic fabrication. Additionally, the images of innovative substrate materials like nanocellulose paper, have further activated the performance of paper-based analytical microfluidic devices.Currently, one of the experimental challenges we face is laser-cutting processing, which limited most wide to 200 micrometers and impacts microchannel accuracy. To overcome this, future research will explore nano-3D printing for MoS, aiming to achieve a higher precision level in nanopaper-based analytic microfluidic devices. In contrast, to establish the techniques for microchannel fabrication on nanocellulose paper, such as 3D printing, spray coating, or manual cutting and assembly, our method offers a distinct advantage.It combines simplicity with superior accuracy, ensuring user friendliness, cost effectiveness, time efficiency, and the attunement of micrometer-level macro channels. Our findings provide a new, accurate and simple method to fabricate microchannels on nanopaper and eventually for making nanopaper-based microfluidic devices. In the big picture, with this method, the existing paper substrate nanopaper can be easily used in the field of microfluidics.

A unique method for creating microchannel patterns on nanopaper has been devised utilizing the practical plastic micro-molds through the convenient microembossing technique. Notably, this method accomplishes microchannel patterning at a scale as small as 200 &#181;m, which represents a fourfold improvement compared to existing methods32,33,34. After fine-tuning the patterning parameters, the provided guidelines exhibit excellent...

Watch this Scientific Journal Video about Revolutionizing Microfluidics Through Microchannel Fabrication on Nanopaper at JoVE.com

This protocol describes a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanofibrillated cellulose paper, achieving a minimum width of 200 &#181;m.

Nanopaper, derived from nanofibrillated cellulose, has generated considerable interest as a promising material for microfluidic applications. Its appeal ...

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Research

JoVE Journal

Engineering

Nano-PAD Fabrication Using Micro-Embossing

To begin, laser cut the 500-micrometer-thick polytetrafluoroethylene or PTFE films using the laser cutting machine to generate the microchannel mold. Disperse four grams of TEMPO-oxidized nanofibrillated cellulose gel in distilled water. Stir the suspension heavily at 120.8 G for 30 minutes until no cellulose flock is seen.Vacuum filter the clear suspension to obtain a nanopaper gel. Next, place the PTFE mold on the surface of the nanopaper gel. Use a hot press to emboss the gel for 10 minutes under 750 kilopascals pressure at 50 degrees Celsius.To release the mold, peel off an additional layer of filter nanopaper gel from the filter membrane. Attach the peeled layer on top of the embossed nanopaper gel and stack the two layers to create a hollow microchannel structure. Dry the stacked layers in a drying oven at 75 degrees Celsius for 30 minutes.Next, prepare the Nano-PADs with straight and curved channels. Add red and blue droplets in the inlet zone simultaneously, allowing automatic channel flowthrough. Microchannel patterning at 200 micrometers was possible with micro-embossing.

1.0K Views.  Xi’an Jiaotong - Liverpool University.  This protocol describes a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanofibrillated cellulose paper, achieving a minimum width of 200 µm.

Micro-Embossed Fabrication of Nanocellulose Paper-Based Analytical Devices

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