The research is supported by the National Natural Science Foundation of China (Grant No. 61974004 and 61931018), as well as the National Key R&#38;D Program of China (Grant No. 2021YFB3200100). The study acknowledges the Electron Microscopy Laboratory of Peking University for providing access to electron microscopes. Additionally, the research extends thanks to Ying Cui and the School of Earth and Space Science of Peking University for their assistance in Raman measurements.

1. Synthesis of nanoparticles
<ol>
	<li>Preparation of Silver nitrate solution
	<ol>
		<li>Using a precision weighing balance, measure 0.0017 g of AR-grade silver nitrate (AgNO3, see Table of Materials) and add it to 10 mL of deionized (DI) water. Stir the mixture to create a 10-3 mol/L AgNO3 solution.</li>
	</ol>
	</li>
	<li>Preparation of the silver-ammonia complex
	<ol>
		<li>Take 1 mL of AR-grade ammonia water (NH3.H2O, see Table of Materials) using a syringe, and add it drop by drop into the silver nitrate solution while stirring. Stop the dropwise addition when the solution becomes clear.</li>
	</ol>
	</li>
	<li>Preparation of the glucose solution
	<ol>
		<li>Using a precise weighing balance, measure 0.36 g of AR-grade glucose powder (see Table of Materials) and add it to 10 mL of DI water. Stir the mixture thoroughly to create a 0.2 M glucose solution.</li>
	</ol>
	</li>
	<li>Synthesis of silver nanoparticles (AgNPs)
	<ol>
		<li>Employ a pipette gun to add 30 &#181;L of the silver-ammonia complex (prepared in step 1.2) to the glucose solution (prepared in step 1.3) at intervals of 30 min. Repeat this process 4-6 times while stirring until the solution turns yellow.</li>
	</ol>
	</li>
</ol>
2. Preparation of flexible substrates
<ol>
	<li>Preparation of PDMS substrate
	<ol>
		<li>To synthesize the PDMS substrate, take approximately 5 g of PDMS A solution and add B solution (from a commercially available kit, see Table of Materials) at a ratio of 1:10.</li>
		<li>Stir and thoroughly mix the PDMS A and B solutions.</li>
		<li>Transfer the mixed PDMS into a square dish and then bake it in an 80 &#176;C oven for 2 h.</li>
		<li>After curing through the above process, use a scalpel to cut the PDMS along the dark grid of the Petri dish, creating small PDMS cubes with dimensions of approximately 1 cm x 1 cm.</li>
	</ol>
	</li>
	<li>Surface modification
	<ol>
		<li>Subject the aforementioned small PDMS pieces to plasma treatment. Use a hand-held plasma processor (see Table of Materials) and move it back and forth approximately 5-10 cm above the PDMS surface to perform surface plasma treatment.</li>
		<li>Utilize the plasma processor to modify the surface, inducing the formation of hydroxyl groups on the PDMS surface, rendering it hydrophilic27.</li>
	</ol>
	</li>
	<li>Modification with APTES
	<ol>
		<li>Prepare a 10% APTES solution (see Table of Materials).</li>
		<li>Immerse the surface-modified PDMS obtained in step 2.2 into the APTES solution and allow it to sit for 10 h. This allows APTES to bind with the hydroxyl groups on the PDMS surface.</li>
	</ol>
	</li>
	<li>Self-assembly of AgNPs
	<ol>
		<li>Immerse the PDMS substrate obtained in step 2.3 into the AgNPs solution synthesized in step 1.4 for 10 h. This self-assembles the AgNPs onto the PDMS substrate, creating the final flexible SERS detection substrate.</li>
	</ol>
	</li>
</ol>

Fabrication of Flexible Substrate for SERS Detection of R6G and Thiram

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The authors declare no conflicts of interest.

In this study, a flexible SERS substrate was introduced, which bonded AgNPs to PDMS through chemical modification and achieved excellent performance. During particle synthesis, specifically in the silver ammonia complex synthesis (step 1.2), the color of the solution plays a crucial role. Adding too much ammonia water dropwise can adversely affect AgNPs synthesis quality, potentially leading to unsuccessful detection results. Attention should be paid to substrate modification (step 2.2) during the synthesis process; otherwise, AgNPs may not bond properly to PDMS, resulting in weakened detection performance.
In practical preparations, the SERS substrate&#39;s detection performance may exhibit instability22. This can be optimized by changing the solvent of the substance. For example, using acetonitrile as a solvent for Thiram yields better results than using ethanol. Additionally, the quality of Thiram can impact the detected SERS signal, emphasizing the importance of ensuring that the reagents used are within their expiration dates during detection.
Compared to other studies36,37,38, the SERS flexible substrate&#39;s detection method proposed in this study is straightforward. AgNPs can be easily synthesized through a simple method, avoiding the need for complex experimental conditions and environments, as well as intricate fabrication processes. The substrate is environmentally friendly and does not introduce harmful pollutants. However, it should be noted that due to the glucose layer around AgNPs, it can weaken the enhancement effect of silver particles, suggesting that further improvement in the Enhancement Factor (EF) of the SERS substrate is necessary. The SERS flexible substrate, prepared using the method in this study, also requires further exploration in the detection of biomolecules.
The flexible SERS substrate proposed in this study demonstrates applicability in real-life scenarios, enriching the methods for pesticide residue detection and carrying significant implications. Furthermore, in future applications, the flexible SERS substrate holds great potential for biomedical applications.

Surface-Enhanced Raman Scattering (SERS), as a type of Raman scattering, offers the advantages of high sensitivity and gentle detection conditions, and can even achieve single molecule detection1,2,3,4. Metal nanostructures, such as gold and silver, are typically used as SERS substrates to enable substance detection5,6. Electromagnetic coupling enhancement on nanostructured surfaces plays a significant role in SERS applications. Metallic nanostructures with varying sizes, shapes, interparticle distances, and compositions can aggregate to create numerous &#34;hotspots&#34; generating intense electromagnetic fields due to localized surface plasmon resonances7,8. Many studies have developed metal nanoparticles with different morphologies as SERS substrates, demonstrating their effectiveness in achieving SERS enhancement9,10.
Flexible SERS substrates find wide applications, with nanostructures capable of producing SERS effects deposited on flexible substrates to facilitate direct detection on curved surfaces. Flexible SERS substrates are employed for detecting and collecting analytes on irregular, non-planar, or curved surfaces. Common flexible SERS substrates include fibers, polymer films, and graphene oxide films11,12,13,14. Among them, polydimethylsiloxane (PDMS) is one of the most widely used polymer materials and offers advantages such as high transparency, high tensile strength, chemical stability, non-toxicity, and adhesion15,16,17. PDMS has a low Raman cross-section, making its impact on the Raman signal negligible18. Since the PDMS prepolymer is in liquid form, it can be cured by heat or light, providing a high degree of controllability and convenience. PDMS-based SERS substrates are relatively common flexible SERS substrates, having been used in previous studies to embed various metal nanoparticles for detecting different biochemical substances with exemplary performance19,20.
In the preparation of SERS substrates, the fabrication of nanogap structures is crucial. Physical deposition technology offers advantages like high scalability, uniformity, and reproducibility but typically requires good vacuum conditions and specialized equipment, limiting its practical applications21. Additionally, fabricating nanostructures at the few-nanometer scale remains challenging with conventional deposition techniques22. Consequently, nanoparticles synthesized through chemical methods can be adsorbed onto flexible transparent films through various interactions, facilitating the self-assembly of metallic structures at the nanoscale. To ensure successful adsorption, interactions can be adjusted by physically or chemically modifying the film surface to alter its surface hydrophilicity23. Silver nanoparticles, compared to gold nanoparticles, exhibit better SERS performance, but their instability, particularly their susceptibility to oxidation in air, results in a rapid decrease in the SERS Enhancement Factor (EF), affecting substrate performance24. Hence, it is essential to develop a stable particle method.
The presence of pesticide residues has garnered significant attention, creating a pressing need for robust methods capable of rapidly detecting and identifying various classes of hazardous chemicals in food in the field25,26. Flexible SERS substrates offer unique advantages in practical applications, particularly in the realm of food safety. This article introduces a method for preparing a flexible SERS substrate by bonding synthesized glucose-coated silver nanoparticles (AgNPs) onto a PDMS substrate (Figure 1). The presence of glucose protects the AgNPs, mitigating silver oxidation in the air. The substrate demonstrates excellent detection performance, capable of detecting Rhodamine 6G (R6G) as low as 10-10 M and pesticide Thiram as low as 10-8 M, with good uniformity. Moreover, the flexible substrate can be employed for detection through bonding and sampling, with numerous potential application scenarios.

<table><tbody><tr><td>Ammonia (NH&lt;sub&gt;3.&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O, 25%)</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>APTES (98%)</td><td>Beyotime</td><td>ST1087</td><td></td></tr><tr><td>BD-20AC Laboratory Chrona Treater</td><td>Electro-Technic Products Inc.</td><td>12051A</td><td></td></tr><tr><td>D-glucose</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>Environmental Scanning electron microscope (ESEM)</td><td>FEI</td><td>QUANTA 250</td><td></td></tr><tr><td>Raman microscope</td><td>Horiba JY</td><td>LabRAM HR Evolution</td><td></td></tr><tr><td>Rhodamine 6G</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>Silicone Elastomer Base and Silicone Elastomer Curing Agent</td><td>Dow Corning Corporation</td><td>SYLGARD 184</td><td></td></tr><tr><td>Silver nitrate</td><td>Beijing Chemical Works</td><td></td><td></td></tr><tr><td>Thiram (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;, 99.9%)</td><td>Beijing Chemical Works</td><td></td><td></td></tr></tbody></table>

fabrication of polydimethylsiloxane (pdms)-based flexible surface-enhanced raman scattering (sers) substrate for ultrasensitive detection

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) were synthesized through a complexation reaction involving silver nitrate (AgNO3) and ammonia, followed by reduction using glucose. The resulting AgNPs exhibited a uniform size distribution ranging from 20 nm to 50 nm. Subsequently, 3-aminopropyl triethoxysilane (APTES) was employed to modify a PDMS substrate that had been surface-treated with oxygen plasma. This process facilitated the self-assembly of AgNPs onto the substrate. A systematic evaluation of the impact of various experimental conditions on substrate performance led to the development of a SERS substrate with excellent performance and an Enhanced Factor (EF). Utilizing this substrate, impressive detection limits of 10-10 M for R6G (Rhodamine 6G) and 10-8 M for Thiram were achieved. The substrate was successfully employed for detecting pesticide residues on apples, yielding highly satisfactory results. The flexible SERS substrate demonstrates great potential for real-world applications, including detection in complex scenarios.

In this study, a flexible SERS substrate composed of synthetic AgNPs wrapped in glucose and self-assembled on PDMS using APTES was developed, achieving excellent detection performance for practical pesticide detection applications. The detection limits for R6G and Thiram were both reached at 10-10 M and 10-8 M, respectively, with an Enhancement Factor (EF) of 1 x 105. Furthermore, the substrate demonstrated uniformity.
The AgNPs wrapped in glucose were synthesized using an improved Tollens method28,29. This AgNPs assembly not only generated a strong SERS signal but also effectively shielded the silver in the AgNPs from oxidation, preserving detection performance. From the Environmental Scanning Electron Microscopy (ESEM) images in Figure 2, the synthesized particles appeared relatively uniform, with most having diameters between 40-50 nm. The outer layer of the AgNPs was enveloped by a glucose layer. This structure provided a dielectric layer for the AgNPs&#39; outer layer and shielded the silver particles from oxidation upon exposure to air, preserving SERS performance.
It is evident that a strong enhanced electric field forms between the gaps of the AgNPs, serving as the primary cause of the SERS signal. Therefore, the substrate is densely immobilized with AgNPs on flexible substrates to achieve enhanced performance (Figure 3). The self-assembled SERS flexible substrate developed in this study is simple, of high quality, and free from toxic or harmful substances, making it environmentally friendly.
The SERS flexible substrate prepared in this study exhibited excellent detection performance. To evaluate a SERS substrate, the critical aspect is its detection capability. Here, the Enhancement Factor (EF) was defined to assess the enhancement performance of the substrate, and R6G (see Table of Materials) was used to determine the detection limit. The EF was described by30:
EF = (ISERS / IRaman) &#215; (NRaman / NSERS)
The peak positions31 of R6G and their corresponding values are presented in Table 1.
In this study, the Raman spectrum was obtained using a 633 nm laser with 10x and 50x objectives. The integration time was set at 10 s for spectrum acquisition during measurement, with the incident laser power at 3.7 mW. By adding 30 &#181;L of R6G solutions with varying concentrations onto the substrate and observing the Raman signal through direct detection, Figure 4 illustrates that the substrate exhibited excellent detection capability, reaching a detection limit of 10-10 M for R6G, indicative of strong detection performance. Subsequently, using 10-5 M R6G as the test probe, the Enhancement Factor (EF) of the substrate was calculated to be 1 x 105 (the calculation process is detailed in Supplementary File 1), demonstrating a notable enhancement effect (Figure 5).
The flexible SERS substrate enabled the detection of pesticides. Thiram, a widely used dithiocarbamate (DTC) pesticide in fruit and vegetable cultivation, aims to control fungal diseases and prevent deterioration during storage and transport32. However, repeated exposure or ingestion of Thiram residues may result in health issues such as lethargy, muscle tone loss, and severe fetal malformations33,34. Therefore, achieving trace-level Thiram detection on the surfaces of fruits and vegetables is crucial. The Raman peaks35 of Thiram and their causes are outlined in Table 2.
Various concentrations of Thiram were applied to the flexible substrate to assess its detection performance. Figure 6 demonstrates that for Thiram detection, its three primary characteristic peaks are clear, and the detection limit is reached at 10-8 M.
The flexible substrate enabled practical detections. In everyday life, pesticide residues sometimes persist on the surfaces of fruits. Consumption of unwashed fruits may pose health risks. In this study, the flexible SERS substrate was applied using a &#34;paste and peel-off&#34; method, attaching the substrate to the surface of an apple and then removing it for inspection.
Figure 7&#160;illustrates that under this method, the detection of 10-7 M Thiram was achieved, with relatively clear spectral lines. Thus, the flexible SERS substrate prepared can facilitate the &#34;paste and peel-off&#34; detection method, effectively identifying pesticide residues on fruit surfaces, and offering valuable practical applications.
The flexible SERS substrate presented in this study not only exhibited remarkable detection performance but also offered practical application scenarios.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65595/65595fig01.jpg" /> 
Figure 1: Schematic design of the PDMS flexible SERS substrate. Illustration depicting the design of the PDMS (Polydimethylsiloxane) flexible substrate used for Surface-Enhanced Raman Scattering (SERS) experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65595/65595fig02.jpg" /> 
Figure 2: ESEM image of synthesized AgNPs. Environment Scanning Electron Microscopy (ESEM) image showing the synthesized AgNPs (silver nanoparticles). The scale bar in the image is 2 &#181;m, and the diameter of the AgNPs ranges from approximately 20 nm to 50 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65595/65595fig03.jpg" /> 
Figure 3: Simulation of AgNPs. Simulation showing AgNPs (silver nanoparticles) with significant localized field enhancement occurring in the gap between the particles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65595/65595fig04.jpg" /> 
Figure 4: SERS signals of different R6G concentrations. Surface-enhanced Raman Scattering (SERS) signals obtained for various concentrations of R6G (Rhodamine 6G). The peak positions in the figure align with those in Table 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65595/65595fig05.jpg" /> 
Figure 5: SERS signals of R6G on the flexible substrate. Surface-enhanced Raman Scattering (SERS) signals of R6G (Rhodamine 6G) collected from 10 random points on the flexible substrate to demonstrate uniformity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65595/65595fig06.jpg" /> 
Figure 6: SERS signals of different Thiram concentrations. Surface-enhanced Raman Scattering (SERS) signals obtained for various concentrations of Thiram. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65595/65595fig07.jpg" /> 
Figure 7: SERS signals of Thiram on the fruit surface. Surface-enhanced Raman Scattering (SERS) signals of Thiram obtained from the surface of an apple using the &#34;paste and peel-off&#34; method. The detection limit reached 10-7 M of Thiram. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak position (cm-1)</td>
			<td>Assignment</td>
		</tr>
		<tr>
			<td>612</td>
			<td>C-C-C in-plane bending vibration</td>
		</tr>
		<tr>
			<td>774</td>
			<td>C-H stretching</td>
		</tr>
		<tr>
			<td>1127</td>
			<td>C-H in-plane bending vibration</td>
		</tr>
		<tr>
			<td>1180</td>
			<td>C-H and N-H bending vibration</td>
		</tr>
		<tr>
			<td>1310</td>
			<td>C=C stretching</td>
		</tr>
		<tr>
			<td>1364</td>
			<td>Stretching vibration of the C-C bond</td>
		</tr>
		<tr>
			<td>1509</td>
			<td>Stretching vibration of the C-C bond</td>
		</tr>
		<tr>
			<td>1574</td>
			<td>Stretching vibration of the C=O bond</td>
		</tr>
		<tr>
			<td>1647</td>
			<td>Stretching vibration of the C-C bond</td>
		</tr>
	</tbody>
</table>
Table 1: Raman shift and frequency mode assignment in R6G SERS spectrum. Table listing Raman shift values and their corresponding frequency mode assignments in the Surface-Enhanced Raman Scattering (SERS) spectrum of R6G (Rhodamine 6G).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak position (cm-1)</td>
			<td>Assignment</td>
		</tr>
		<tr>
			<td>440</td>
			<td>CH3-N-C deformation (&#948; (CH3-N-C)), C=S stretching (&#965;(C=S))</td>
		</tr>
		<tr>
			<td>549</td>
			<td>S-S symmetric stretching (&#965;s (S-S))</td>
		</tr>
		<tr>
			<td>928</td>
			<td>C=S stretching (&#965; (C=S)), C-N stretching (&#965; (CH3-N))</td>
		</tr>
		<tr>
			<td>1136</td>
			<td>C-N stretching (&#965; (C-N)), the rocking CH3 modes (&#961;(CH3))</td>
		</tr>
		<tr>
			<td>1388</td>
			<td>C-N stretching (&#965; (C-N)), CH3 symmetric deformation (&#965;(C=S))</td>
		</tr>
	</tbody>
</table>
Table 2: Raman shift and frequency mode assignment in Thiram SERS spectrum. Table listing Raman shift values and their corresponding frequency mode assignments in the Surface-Enhanced Raman Scattering (SERS) spectrum of Thiram.
Supplementary File 1: Calculation of Enhancement Factor (ER). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65595/65595_Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Development and Application of SERS Flexible Substrates Using Synthesized AgNPs at JoVE.com

Author Spotlight: Development and Application of SERS Flexible Substrates Using Synthesized AgNPs 

This protocol describes a fabrication method for a flexible substrate for surface-enhanced Raman scattering. This method has been used in the successful detection of low concentrations of R6G and Thiram.

Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) ...

fabrication-polydimethylsiloxane-pdms-based-flexible-surface-enhanced

Nanyang Technological University

Research

JoVE Journal

Engineering

2.1K Views.  Peking University. Dit protocol beschrijft het voorbereiden van flexibele SER-substraten en het toepassen ervan op daadwerkelijke detectiescenario's. Het flexibele SERS-substraat dat door dit protocol wordt aangeboden, is milieuvriendelijk en behaalt goede detectieprestaties. De zilveren nanodeeltjes kunnen gemakkelijk worden gesynthetiseerd door eenvoudige vaten, en vereisen geen ingewikkelde experimentele omstandigheden en omgeving.In ons laboratorium zijn we van plan ons te concentreren op het onderzo...

Video: Auteur in de schijnwerpers: Ontwikkeling en toepassing van flexibele SER-substraten met behulp van gesynthetiseerde AgNP's 

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Bioengineering

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

Genetics

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Chemistry

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Fabrication of Flexible Image Sensor Based on Lateral NIPIN Phototransistors

Flexible photodetectors have been intensely studied for the use of curved image sensors, which are a crucial component in bio-inspired imaging systems, but several challenging points remain, such as a low absorption efficiency due to a thin active layer and low flexibility. We present an advanced method to fabricate a flexible phototransistor array with an improved electrical performance. The outstanding electrical performance is driven by a low dark current owing to deep impurity doping. Stretchable and flexible metal interconnectors simultaneously offer electrical and mechanical stabilities in a highly deformed state. The protocol explicitly describes the fabrication process of the phototransistor using a thin silicon membrane. By measuring I-V characteristics of the completed device in deformed states, we demonstrate that this approach improves the mechanical and electrical stabilities of the phototransistor array. We expect that this approach to a flexible phototransistor can be widely used for the applications of not only next-generation imaging systems/optoelectronics but also wearable devices such as tactile/pressure/temperature sensors and health monitors.

We present a detailed method to fabricate a deformable lateral NIPIN phototransistor array for curved image sensors. The phototransistor array with an open mesh form, which is composed of thin silicon islands and stretchable metal interconnectors, provides flexibility and stretchability. The parameter analyzer characterizes the electrical property of the fabricated phototransistor.

Flexible photodetectors have been intensely studied for the use of curved image sensors, which are a crucial component in bio-inspired imaging systems, ...

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Sensitivity Enhancement of Soft Capacitive Pressure Sensors Using a Solvent Evaporation-Based Porosity Control Technique

Soft pressure sensors play a significant role in developing &#34;man-machine&#34; tactile sensation in soft robotics and haptic interfaces. Specifically, capacitive sensors with micro-structured polymer matrices have been explored with considerable effort because of their high sensitivity, wide linearity range, and fast response time. However, the improvement of the sensing performance often relies on the structural design of the dielectric layer, which requires sophisticated microfabrication facilities. This article reports a simple and low-cost method to fabricate porous capacitive pressure sensors with improved sensitivity using the solvent evaporation-based method to tune the porosity. The sensor consists of a porous polydimethylsiloxane (PDMS) dielectric layer bonded with top and bottom electrodes made of elastic conductive polymer composites (ECPCs). The electrodes were prepared by scrape-coating carbon nanotubes (CNTs)-doped PDMS conductive slurry into mold-patterned PDMS films. To optimize the porosity of the dielectric layer for enhanced sensing performance, the PDMS solution was diluted with toluene of different mass fractions instead of filtering or grinding the sugar pore-forming agent (PFA) into different sizes. The evaporation of the toluene solvent allowed the fast fabrication of a porous dielectric layer with controllable porosities. It was confirmed that the sensitivity could be enhanced more two-fold when the toluene to PDMS ratio was increased from 1:8 to 1:1. The research proposed in this work enables a low-cost method of fabricating fully integrated bionic soft robotic grippers with soft sensory mechanoreceptors of tunable sensor parameters.

A simple and cost-efficient fabrication method based on the solvent evaporation technique is presented to optimize the performance of a soft capacitive pressure sensor, which is enabled by porosity control in the dielectric layer using different mass ratios of the molding PDMS/toluene solution.

Soft pressure sensors play a significant role in developing &#34;man-machine&#34; tactile sensation in soft robotics and haptic interfaces. Specifically, ...

Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection

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