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...

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 ...

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

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

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. 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 ...

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 ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...