Name: dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation
Uploaded: 2017-09-19T12:30:00
Description: Watch this Scientific Journal Video about Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation at JoVE.com

The authors would like to thank the German Research Foundation (DFG) for partially funding this work under Grant Numbers UR 70/10-01 and UR 70/12-01.

1. Fabrication of the Microfluidic Biosensor Using DFR Technology
<ol>
	<li>Preparation of the PI wafers.
	<ol>
		<li>Cut a PI substrate into 6-in round wafers. Put the PI wafer in an oven at 120 &#176;C for roughly 1 h for a dehydration bake.</li>
	</ol>
	</li>
	<li>First photolithography step for the lift-off process.
	<ol>
		<li>Program the spin-coater to a 30-s spinning time at 3,000 rpm, with an acceleration of 2,000 rpm/s. Place the PI wafer on the spin-coater and fix it, applyin...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+point-of-care+testing+-+xPOCT.">Multiplexed point-of-care testing - xPOCT.</a> Trends Biotechnol. 35, (2017).</li><li>Horak, J., Dincer, C., Bakirci, H., Urban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+disposable+dry+film+photoresist-based+microcapillary+immunosensor+chip+for+rapid+detection+of+Epstein-Barr+virus+infection.">A disposable dry film photoresist-based microcapillary immunosensor chip for rapid detection of Epstein-Barr virus infection.</a> Sens Actuators B Chem. 191, 813-820 (2014).</li><li>Jobst, G., Gamp, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+fabrication+of+a+&amp;#34;lab+on+chip&amp;#34;+from+photoresist+material+for+medical+diagnostic+applications.">Method for the fabrication of a &amp;#34;lab on chip&amp;#34; from photoresist material for medical diagnostic applications.</a> US patent. , (2010).</li><li>Kling, A., Dincer, C., Armbrecht, L., Horak, J., Kieninger, J., Urban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+microfluidic+platform+for+simultaneous+multi-analyte+detection.">Electrochemical microfluidic platform for simultaneous multi-analyte detection.</a> Procedia Eng. 120, 916-919 (2015).</li><li>Armbrecht, L., Dincer, C., Kling, A., Horak, J., Kieninger, J., Urban, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+magnetic+bead+chains+for+sensitivity+enhancement+of+microfluidic+electrochemical+biosensor+platforms.">Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms.</a> Lab Chip. 15, 4314-4321 (2015).</li><li>Dincer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+miniaturization+of+microfluidic+biosensor+platforms+using+the+stop-flow+technique.">Designed miniaturization of microfluidic biosensor platforms using the stop-flow technique.</a> Analyst. 141, 6073-6079 (2016).</li><li>Weltin, A., Kieninger, J., Enderle, B., Gellner, A. 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The protocol presented here for the fabrication of a microfluidic electrochemical biosensor enables the development of a low-cost, compact, and easy-to-use platform for the detection of biomolecules. Depending on the assay used afterwards on the biosensor, several different biomarkers can be detected. This makes the platform very versatile and provides broad access to various fields of applications, from standard diagnostic tests (e.g., determining the presence of specific diseases at the doctor&#39;s office) to...

Over the past two decades, diagnostic applications have become elementary for in-depth studies on the development of global public health. Traditionally, laboratory diagnostic tools are used for the detection of diseases. Even though they still play a key role in the diagnosis of diseases, point-of-care testing (POCT) performed near the patient or by the patient himself has become more and more commonplace in recent years. Especially in such cases that require immediate treatment, such as acute myocardial infarction or diabetes monitoring, the rapid confirmation of a clinical finding is essential. Hence, there is a growing need for POCT devices that can be operated by non-experts and that are concurrently capable of performing precise in vitro diagnostic tests in a short time1,2,3,4.
Remarkable improvements have already been achieved in the field of POCT. However, there are still many challenges to overcome5,6,7,8. For a POCT platform to be successfully launched to the market and to be competitive with laboratory diagnostics, the device must strictly fulfill the following requirements: (i) provide precise and quantitative test results that are consistent with laboratory findings; (ii) have short sample-to-result times, enabling the immediate treatment of the patient; (iii) feature uncomplicated and easy handling, even when operated by untrained individuals, and require minimized user intervention; and (iv) comprise of a low-cost sensor unit designed for single-use applications. Furthermore, equipment-free diagnostics are favorable, mainly in resource-poor environments3,4,6.
Due to these severe requirements, only two POCT systems based on electrochemical detection (e.g., blood glucose test strips) and on lateral flow immunoassays (e.g., home pregnancy tests) have been successfully launched to the market so far. However, both systems suffer from disadvantages such as poor performance (i.e., blood glucose monitoring has inaccurate test results and lateral flow assays only provide qualitative (positive or negative) measurement results)4,6. These drawbacks of conventional POCT systems have led to an increasing demand on exploring new technologies that offer fast, low-cost, and quantitative detection at the point of care4,5.
To meet these challenges facing POCT devices, DFR technology has been recently employed for the fabrication of disposable and low-cost biosensors9,10,11,12,13,14. Compared to soft and liquid lithographic materials, such as PDMS or SU-8, DFRs present many benefits: they (i) are available in a variety of compositions and thicknesses (from a few microns to several millimeters); (ii) have a very rough surface area, which facilitates adhesion to various materials; (iii) feature excellent thickness uniformity; (iv) offer cheap, facile, and high-throughput fabrication for mass production; (v) are easy to cut with various low-cost tools, like a simple pair of scissors; and (vi) allow for the creation of three-dimensional structures, such as microfluidic channels, by stacking multiple DFR layers on top of each other.
On the other hand, DFRs in general have a relatively poor resolution compared to liquid photoresists, which is mainly caused by the film thickness and by the increased distance between the mask and the DFR due to the protective foil, which additionally enables light scattering. Still, for the manufacturing of integrated microfluidic biosensors, DFRs are highly suitable for low-cost mass production.
Therefore, we present in this work the fabrication and application of a DFR-based electrochemical microfluidic biosensor. The detailed protocol describes each production step of the biosensor platform, the on-chip immobilization of a DNA-based model assay, and its electrochemical readout using the stop-flow technique. This universal platform enables the detection of numerous kinds of biomolecules, using different assay technologies (e.g., genomics, cellomics, and proteomics) or assay formats (e.g., competitive, sandwich, or direct). Based on such a DFR platform, our group previously successfully demonstrated the rapid and sensitive quantification of various analytes, including antibiotics13,15,16 (tetracycline, pristinamycin, and &#223;-lactam antibiotics), troponin I17, and substance P18.

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyralux</td>
			<td>DuPont</td>
			<td>AP8525R</td>
			<td>Used as polyimide substrate</td>
		</tr>
		<tr>
			<td>MA-N 1420</td>
			<td>Micro Resist Technology</td>
			<td>MA-N1420</td>
			<td>Lift-off resit to define the platinum depostion</td>
		</tr>
		<tr>
			<td>Ma-D 533s</td>
			<td>Micro Resist Technology</td>
			<td>MaD533S</td>
			<td>Developer for MA-N1420</td>
		</tr>
		<tr>
			<td>Platinum</td>
			<td>-</td>
			<td>-</td>
			<td>Electrode and contact pad material</td>
		</tr>
		<tr>
			<td>Ma-R 404s</td>
			<td>Micro Resist Technology</td>
			<td>MaR404S</td>
			<td>Remover for MA-N1420</td>
		</tr>
		<tr>
			<td>SU-8 3005</td>
			<td>MicroChem Corp.</td>
			<td>SU-8-3005</td>
			<td>Photoresist to define the electrode area and as insulation</td>
		</tr>
		<tr>
			<td>1-methoxy-2-propanol acetate</td>
			<td>Sigma-Aldrich</td>
			<td>108-65-6</td>
			<td>Developer for SU-8 3005</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>VWR</td>
			<td>8.18766.2500</td>
			<td>Removing of the SU-8 developer</td>
		</tr>
		<tr>
			<td>1020R</td>
			<td>Ultron Systems Inc.</td>
			<td>1020R</td>
			<td>UV sensitive adhesive tape for protection of contact pads</td>
		</tr>
		<tr>
			<td>Arguna S</td>
			<td>Degussa</td>
			<td>1935</td>
			<td>For Silver depostion on reference electrode</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Methrom</td>
			<td>62308.020</td>
			<td>For chloridation of the silver reference electrode</td>
		</tr>
		<tr>
			<td>Pyralux</td>
			<td>DuPont</td>
			<td>PC1025</td>
			<td>Dry film photoresist</td>
		</tr>
		<tr>
			<td>Sodium carbonat</td>
			<td>Fluka</td>
			<td>71352</td>
			<td>Developer for Pyralux PC1025</td>
		</tr>
		<tr>
			<td>Hydrogen chloride</td>
			<td>Sigma-Aldrich</td>
			<td>30720</td>
			<td>To top the development of the DFR</td>
		</tr>
		<tr>
			<td>Teflon AF 1600</td>
			<td>DuPont</td>
			<td>AF1600</td>
			<td>For employing the stopping barrier</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PA104</td>
			<td>Mega Electronics</td>
			<td>-</td>
			<td>Bubble etch tank</td>
		</tr>
		<tr>
			<td>FED 53</td>
			<td>Binder</td>
			<td>9010-0018</td>
			<td>Oven</td>
		</tr>
		<tr>
			<td>SPIN150</td>
			<td>APT</td>
			<td>-</td>
			<td>Spin coater</td>
		</tr>
		<tr>
			<td>Pr&#228;zitherm</td>
			<td>Harry Gestigkeit GmbH</td>
			<td>PZ 28-2</td>
			<td>Hot plate</td>
		</tr>
		<tr>
			<td>Hellas</td>
			<td>Bungard Elektronik</td>
			<td>40000</td>
			<td>Exposure unit</td>
		</tr>
		<tr>
			<td>Tetra30-LF-PC</td>
			<td>Diener</td>
			<td>-</td>
			<td>Plasma unit</td>
		</tr>
		<tr>
			<td>Univex 500</td>
			<td>Leybold</td>
			<td>-</td>
			<td>Physical vapor deposition unit</td>
		</tr>
		<tr>
			<td>Shaker S4</td>
			<td>ELMI</td>
			<td>-</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Sonorex Super 10 P</td>
			<td>Bandelin</td>
			<td>783</td>
			<td>Sonic bath</td>
		</tr>
		<tr>
			<td>6221 DC and AC</td>
			<td>Keithley</td>
			<td>-</td>
			<td>Current source</td>
		</tr>
		<tr>
			<td>HRL 350</td>
			<td>Ozatec</td>
			<td>-</td>
			<td>Laminator unit</td>
		</tr>
		<tr>
			<td>Vaccum pen</td>
			<td>EFD</td>
			<td>-</td>
			<td>Vacuum pen</td>
		</tr>
	</tbody>
</table>

dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

Design and Fabrication of the Microfluidic Biosensor Platform:
The fabrication of the microfluidic biosensor chips is realized on the wafer-level by standard photolithographic techniques employing multiple DFR layers. This fabrication strategy relies on the lamination of developed layers of DFRs on a platinum-patterned PI substrate, forming the microfluidic channels. A short summary depicting the different fabrication steps is give...

Watch this Scientific Journal Video about Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation at JoVE.com

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

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