Name: dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation
Uploaded: 2017-09-19T12:30:00.000+00:00
Duration: 13 min 42 s
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, applying a vacuum. Dispense 2 mL of a resist, enabling the lift-off process, and start the spin-coater program. Remove the wafer from the spin-coater and soft-bake the PI wafer on a hot plate for 2 min at 100 &#176;C.</li>
		<li>Adjust the desired mask for patterning the electrodes on the PI wafer with the naked eye and expose it to 400 mJ/cm2 UV light (365 nm). Place the wafer in a bowl filled with a resist-matching developer on an orbital shaker and let it shake slightly for 1 min.</li>
		<li>After the excess resist is removed, use a shower to rinse the PI wafer with deionized water (DI-water) on a wet bench and dry it afterwards using compressed air.</li>
	</ol>
	</li>
	<li>Deposition of platinum for the formation of the electrodes.
	<ol>
		<li>Use a standard physical vapor deposition process to deposit 200 nm of platinum onto the wafer19.</li>
		<li>Place the wafer in a bowl. Add the matching remover to the bowl and remove the lift-off resist while slightly shaking the wafer on an orbital shaker until all excess platinum is removed. Rinse and dry the PI wafer as described in step 1.2.3.</li>
	</ol>
	</li>
	<li>Second photolithography step: Forming the insulation layer.
	<ol>
		<li>Put the PI wafer in an oven preheated to 120 &#176;C for 5 min to dehydrate it.</li>
		<li>Program the first step of a spin-coater to 5 s of spinning time at 500 rpm. Program the second step for 30 s at 4,000 rpm, with an acceleration of 700 rpm/s.</li>
		<li>Place the PI wafer on the spin-coater and fix it, applying a vacuum. Dispense 4 mL of an appropriate epoxy-based photoresist before starting the spin-coater program.</li>
		<li>Soft-bake the coated wafer for 3 min at 95 &#176;C on a hot plate.</li>
		<li>Adjust the mask for the insulation layer on the wafer using the respective alignment marks and an ocular lens. Expose the wafer to 100 mJ/cm2 UV light (365 nm).</li>
		<li>For a post-exposure bake, place the wafer on a preheated hot plate for 2 min at 95 &#176;C.</li>
		<li>Place the wafer into a bowl and develop the resist with a suitable developer (e.g., 1-methoxy-2-propyl-acetat for 2 min, in the case of SU-8) on an orbital shaker.</li>
		<li>Rinse the PI wafer first with isopropanol and then rinse and dry it as described in step 1.2.3.</li>
		<li>Hard-bake the photoresist in an oven for 3 h at 150 &#176;C.</li>
	</ol>
	</li>
	<li>Plasma-assisted cleaning of the electrodes.
	<ol>
		<li>To remove the photoresist residues, use oxygen plasma with a standard plasma unit. Start the cleaning program, using 200-W low frequency power with 100% O2 flow, a rate of 250 sccm, and a total time of 3 min.</li>
		<li>Place the PI wafer into the plasma chamber, fix the wafer on the grounded plate using glass lids, and start the plasma process.</li>
	</ol>
	</li>
	<li>Silver and silver chloride deposition: Creating the on-chip reference electrode.
	<ol>
		<li>Passivate the platinum contact pads of the wafer with a UV-sensitive adhesive tape. Cut the foil in to 5 mm-wide and 11 cm-long stripes and attach them to the wafer, protecting the parts not to be deposited with silver.</li>
		<li>For the silver deposition, put a sonic bath (35 kHz) into a fume cupboard and insert a container of silver electrolyte solution (100 g/L Ag, pH 12.5) into the bath. Set the sonic bath to room temperature and the power to 10%. 
		Caution: Silver electrolyte solutions are highly toxic and should be handled with extreme care. The fumes should never be inhaled.</li>
		<li>Connect the bulk contact of the reference electrodes to a constant current source. Connect the counter electrode, a silver wire that is immersed in the silver electrolyte solution, to the current source using standard connecting cables.</li>
		<li>Set the current source to DC and to a current density of -4.5 mA/cm2, resulting in a silver deposition rate of approximately 0.3 &#181;m/min. Start the sonic bath and let the current source run for 10 min. Rinse the wafer with DI-water</li>
		<li>For the chlorination of the reference electrode, insert a vessel of 0.1 M potassium chloride (KCl) solution into the sonic bath. Connect the bulk contact of the wafer and a platinum electrode that is immersed in the KCl solution with the constant current source.</li>
		<li>Set the current source to DC and to a current density of +0.6 mA/cm2, resulting in a deposition rate of approximately 0.075 &#181;m/min. Start the sonic bath and let the current source run for 7.5 min.</li>
		<li>Rinse and dry the PI wafer, as described in step 1.2.3.</li>
		<li>Remove the UV-sensitive tape after a UV (365 nm) exposure of 300 mJ/cm2 and rinse and dry the PI wafer, again as described in step 1.2.3.</li>
	</ol>
	</li>
	<li>Third photolithography step: Using DFRs to create the microfluidic channels.
	<ol>
		<li>Cut the needed DFR layers to a size similar to that of the wafer (20 x 20 cm2). Fix the desired mask onto the exposure unit and align the DFR layer onto the mask using the naked eye. Illuminate the resist with 250 mJ/cm2 UV light (365 nm).</li>
		<li>Remove the protective foil of the photoresist and develop the resist for roughly 2 min (depending on the structures) in a 1% sodium carbonate (Na2CO3) solution using a standard sonic bath (35 kHz) preheated to 42 &#176;C and a sonic power of 100%. To stop the reaction immediately, shake the resist for 1 min in a 1% HCl bath on an orbital shaker. Rinse and dry each DFR layer, as described in step 1.2.3. 
		NOTE: In total, four DFR layers need to be processed as mentioned in step 1.7: one channel layer, one cover layer, and two backside layers (preventing the biosensor from bending at the end).</li>
	</ol>
	</li>
	<li>Lamination of the DFR layers on the PI substrate.
	<ol>
		<li>Place the PI wafer onto a transparency overhead foil and fix it using standard adhesive tape. Adjust the channel DFR layer on the PI wafer under a microscope, using the respective alignment structures.</li>
		<li>For the lamination, use a standard hot roll laminator and preheat the top roll to 100 &#176;C and the bottom roll to 60 &#176;C. Set the pressure to 3 bar, with a forward speed of 0.3 m/min. Place the wafer with the fixed DFR layer in the middle of the laminator and start it; the DFR is pushed through the laminator. Repeat the step after rotating the wafer by 180&#176;.</li>
		<li>To prevent the bending of the biosensor, laminate the two developed backside layers onto the wafer following steps 1.8.1-1.8.2.</li>
	</ol>
	</li>
	<li>Employing a hydrophobic stopping barrier and sealing the chip.
	<ol>
		<li>Remove the protective layer from the front side of the DFR channel layer by placing standard adhesive tape at one end of the DFR and pulling it upwards. Use a hand dispenser with 0.004-in tubes to dispense small droplets of dissolved polytetrafluoroethylene into the wells of the insulation layer. To seal the microfluidic chip, laminate the cover DFR layer onto the channel layer, as described in step 1.8.</li>
	</ol>
	</li>
	<li>Hard-baking the microfluidic biosensor.
	<ol>
		<li>Remove all protective foils on the cover and backside DFR layers. Cut the biosensors into strips using an ordinary pair of scissors. Cure the chips in an oven at 160 &#176;C for 3 h.</li>
	</ol>
	</li>
</ol>
2. On-chip Assay Immobilization Procedure
<ol>
	<li>Adsorption of avidin in the immobilization area of the channel.
	<ol>
		<li>Prepare a 100 &#181;g/mL avidin solution in 10 mM phosphate-buffered saline (PBS) at a pH of 7.4.</li>
		<li>Dispense 2 &#181;L of the avidin solution onto the inlet of the biosensor. 
		NOTE: The channel is filled by capillary forces until the fluid reaches the hydrophobic stopping barrier.</li>
		<li>To ensure that the fluidic keeps stopping at the barrier during the whole incubation time, dispense 2 &#181;L of DI-water on the outlet of the chip. Incubate the chip at 25 &#176;C for 1 h in a closed container.</li>
		<li>Remove excess reagents through the inlet of the chip by applying a vacuum. Wash the channel with 50 &#181;L of wash buffer (PBS with 0.05% Tween 20), dispensed on the outlet while the vacuum is being applied. Dry the channel for 30 s while the vacuum is being applied.</li>
	</ol>
	</li>
	<li>Blocking the channel surface to inhibit unspecific binding.
	<ol>
		<li>Prepare a 1% bovine serum albumin (BSA) solution in 10 mM PBS at a pH of 7.4.</li>
		<li>Pipette 2 &#181;L of the 1% BSA solution on the inlet and 2 &#181;L of DI-water on the outlet of the channel. Incubate the chip at 25 &#176;C for 1 h in a closed container. Remove excess reagents and dry the channel as described in step 2.1.4.</li>
	</ol>
	</li>
	<li>Incubation of DNA oligos labeled with biotin and 6-FAM.
	<ol>
		<li>Prepare different concentrations (0.001-5 &#181;M) of the DNA solution in 10 mM PBS at a pH of 7.4.</li>
		<li>Dispense 2 &#181;L of one concentration of the DNA solution on the inlet and 2 &#181;L of DI-water on the outlet. Incubate the chip at 25 &#176;C for 15 min in a closed container.</li>
		<li>Repeat step 2.3.2 for the other DNA concentrations using additional biosensor chips.</li>
		<li>Remove excess reagents and dry the channel as described in step 2.1.4.</li>
	</ol>
	</li>
	<li>Immobilization of GOx-labeled 6-FAM antibodies.
	<ol>
		<li>Formulate a 5 &#181;g/mL concentration of 6-FAM antibodies in 10 mM PBS at a pH of 7.4.</li>
		<li>Introduce 2 &#181;L of the antibody solution to the inlet and 2 &#181;L of DI-water to the outlet ofthe channel. Incubate the labeled antibodies at 25 &#176;C for 15 min in a closed container. Remove the excess reagents, as described in step 2.1.4.</li>
	</ol>
	</li>
</ol>
3. Amperometric Signal Detection Using the Stop-flow Technique
<ol>
	<li>Preparation of the substrate solution for electrochemical detection.
	<ol>
		<li>Formulate a 40-mM glucose solution in 0.1 M PBS at a pH of 7.4 in ultra-pure water.</li>
		<li>For the preliminary treatment, prepare a 0.1 M PBS solution at a pH of 7.4 in ultra-pure water.</li>
	</ol>
	</li>
	<li>Preliminary treatment of the working electrodes.
	<ol>
		<li>Use the custom-made chip holder to allow for the easy placement of the chip and a fluidic and electric connection.</li>
		<li>Electrically connect the biosensor to the potentiostat. Ensure the fluidic contact of the microfluidic channel to a syringe pump and to the reagent reservoir using the custom-made adapter and tubes. Start the laptop that is connected to the potentiostat and start the syringe pump controller, controlling the fluidic flow through the chip.</li>
		<li>Start the syringe pump manually, with 0.1 M PBS at a flow rate of 20 &#181;L/min. At the working electrode versus the on-chip reference electrode, apply an alternating voltage of 0.8 and -0.05 V for 5 s each for 30 cycles. Following this, oxidize the working electrode by applying a voltage of 0.8 V for 60 s.</li>
	</ol>
	</li>
	<li>Assay readout using the stop-flow technique.
	<ol>
		<li>To start the assay signal readout, wait until the measured current signal stabilizes. Stop the syringe pump and switch the reagent from 0.1 M PBS to the 40-mM glucose solution and start the software at the syringe pump controller; it will automatically stop the flow for 1, 2, or 5 min and will then restart the flow again. Observe the resulting current peak. 
		NOTE: For the analysis of the data, either the current peak or the charge of the peak can be considered, as described in the model of an electrochemical signal readout (Figure 2).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 point-of-care applications (e.g., the therapeutic drug monitoring of a patient for individualized drug therapy). Especially in point-of-care diagnostics, miniaturized biosensors have many advantages over conventional methods, which usually require extensive laboratory equipment, specialized employees, and large reagent and sample volumes and have long turnaround times.
The fabrication of this biosensor requires strictly following the protocol; otherwise, manufacturing problems can occur. One of the most often observed issues is the misalignment of the different layers. This can be easily solved by using a more advanced apparatus for the fabrication process, which allows for the easier and more precise alignment of the different layers.
The DFR technology offers the possibility of fast and low-cost fabrication. On the other hand, the technology is limited in terms of resolution. For example, microfluidic channels of very small structures (less than 100 &#181;m) cannot be realized using the protocol presented here. In such a case, the photolithography must be performed by a high-precision mask aligner with glass photomasks. In addition, a too-wide channel can also be problematic, since the channel could bend down, reducing the height of the channel and influencing the microfluidic behavior of the chip.
We believe that DFR technology will be more present in future applications because it can easily be scaled up for commercial use. The mass-production of DFR-based microfluidic sensors will be no hindrance when it hits the market because the technique is well-established in other fields of application (e.g., flexible electronics).
In terms of reducing the complexity of the whole system, our future work will focus on the design and implementation of a disposable microfluidic cartridge that includes the biosensor chip; a waste reservoir; and pre-loaded reagents, such as wash buffer and other assay components. For the amperometric measurement, the delivery of the sample and other reagents can then be provided by pneumatic actuation. This will be performed by the handheld reader and will move through the microfluidic biosensor chip to a waste reservoir.

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>&lt;strong&gt;Material&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;auml;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 given in Figure 1. A single 6-in wafer comprises 130 microfluidic biosensors, each with a dimension of 8 &#215; 10 mm2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56105/56105fig1.jpg" /> 
Figure 1. Graphical illustration of the different fabrication steps of the microfluidic biosensor platform. a) Cut the PI substrate into 6-in round wafers. b) Exposure of a possible spin-coated lift-off resist using the respective mask for platinum patterning. c) Substrate after exposure, post-exposure back, and developing the photoresist. d) Physical vapor deposition of platinum on the substrate. e) Lift-off process to remove the excess photoresist. f) Spin-coating of SU-8, forming an insulation layer. g) O2 plasma process to remove SU-8 residues on the Pt electrodes. h) Galvanic deposition of the Ag/AgCl reference electrode. i) UV exposure and development of different DFR layers. j) Lamination of the DFR layers onto the PI wafer. k) Dispensing solved polytetrafluoroethylene, forming a hydrophobic stopping barrier between the immobilization capillary and the electrochemical cell. l) Final electrochemical microfluidic biosensor. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56105/56105fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Each biosensor consists of one microfluidic channel, separated into two distinct areas by a hydrophobic stopping barrier: an immobilization section and an electrochemical cell, marked in red and blue, respectively, in Figure 2. The immobilization part of the microchannel has a surface volume of 10.34 mm2 and a volume of 580 nL, resulting in a high surface-to volume ratio of 155 cm-1. The electrochemical cell includes an on-chip silver/silver chloride reference electrode and a counter and working platinum electrode. This separation of the immobilization area and the electrochemical readout of the assay prevents any contamination of the electrodes with biomolecules and therefore inhibits electrode fouling. Furthermore, it enables the precise metering of the immobilization reagents by capillary filling.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56105/56105fig2.jpg" /> 
Figure 2. Illustration of the operating principle of the electrochemical microfluidic platform. a) Schematics of a model assay based on avidin. b) Photograph of the microfluidic biosensor showing its main elements, including the counter electrode (CE), the reference electrode (RE), the working electrode (WE), and the stopping barrier (SB). The immobilization area is highlighted in red, and the electrochemical cell is marked in blue. c) Schematic reaction of the oxidation of the produced hydrogen peroxide at the Pt working electrode for amperometric detection inside the electrochemical cell. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56105/56105fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
By employing DFRs for the fabrication of the sensor, the manufacturing process allows for high-throughput on the wafer-level. Therefore, the costs can be kept down to a minimum. The development of all DFR layers is done using simple and low-cost foil masks and a vacuum exposure unit, rather than costly chrome masks and a mask aligner. In addition, the reduction of the necessary clean-room process steps to a minimum cuts the costs for the fabrication even more. In total, the fabrication procedure takes a workload of roughly 10 h, excluding the baking times and the physical vapor deposition of the platinum.
On-chip Assay Incubation:
The on-chip assay incubation is done only by capillary forces. By pipetting drops of different reagents onto the inlet of the microfluidic channel, the fluids are driven through the channel by capillarity action until they reach the hydrophobic stopping barrier. Under steady-state conditions, the reagents are then incubated for a distinct time. This passive system enables the capillary filling of the channel, without the need of any external instrumentation like a syringe pump, and allows for the precise metering of the reagents, as the fluid automatically stops at the stopping barrier. Also, the workflow is consistent with that of a conventional ELISA and it works with high-viscosity liquids like serum or blood.
For the purpose of this experiment, the chip operation is demonstrated by a simple test assay employing the avidin-biotin interaction, as shown in Figure 2. The on-chip assay incubation starts with the adsorption of the avidin to the capillary surface for 1 h, followed by a blocking step with BSA for another 1 h. Subsequently, 6-FAM/biotin-labeled DNA is incubated in the immobilization capillary for 15 min, where it binds to the avidin molecules. Between each incubation step, any unbound biomolecules are removed by a washing step. The wash buffer is applied via the channel outlet using a custom-made vacuum adapter. In the last step, glucose oxidase-labeled antifluorescein antibodies are introduced to the channel for 15 min. After that, the biosensor is ready for the electrochemical readout, using a 40-mM glucose solution as the substrate.
Amperometric Signal Readout Using the Stop-flow Technique:
For the signal readout of the immobilized assay, the enzymes product (here, H2O2), produced in the presence of its substrate, glucose, can be electrochemically detected at the working electrode in the electrochemical cell. To achieve fast detection along with signal amplification, the so-called stop-flow technique is used13. In this method, the flow of the substrate is stopped, which leads to an accumulation of the product inside the capillary. The amount of produced H2O2 therefore depends on the amount of bound glucose oxidase and is dependent on the amount of bound biotinylated DNA. By restarting the flow, the enhanced H2O2 concentration is subsequently flushed through the electrochemical measurement cell, resulting in a current peak signal, as illustrated in Figure 3.
For data analysis, the stop-flow technique offers two different parameters: the maximum height and the charge (i.e., the integral) of the peak signal. Both parameters are directly proportional to the stopping time and can therefore be used for analyzing the data. Considering the data evaluation, when using the peak height, the signal height depends on the maximum H2O2 concentration and thus on its diffusion coefficient and the applied stop time. The longer the stopping time, the higher the measured signal response. For this reason, the gauged peak height remains constant, down to a minimum length of the immobilization capillary. This special feature of the stop-flow technique allows for a drastic decrease in chip dimensions, particularly in the capillary length, while preserving the sensitivity of the sensor.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56105/56105fig3.jpg" /> 
Figure 3. Model of a typically obtained electrochemical signal readout of an enzyme-linked assay using the stop-flow technique. a) A flow of 40-mM glucose solution at a rate of 20 &#181;L/min was applied. During the stop phase (1, 2, or 5 min), the enzyme production of H2O2 continues. By restarting the flow, the accumulated H2O2 is flushed through the electrochemical cell, where the hydrogen peroxide is electrochemically detected. By repeating the measurement several times (error bars; SEM), an on-chip calibration curve b) is obtained. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56105/56105fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

dry-film-photoresist-based-electrochemical-microfluidic-biosensor

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

11.6K Views.  University of Freiburg. 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.

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

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Chemistry

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...