We thank Prof. Paul McEuen at Cornell University for early discussion. The work is supported by the start-up fund provide by the University of Michigan and the National Science Foundation Scalable Nanomanufacturing Program (DMR-1120187). This work used the Lurie Nanofabrication Facility at University of Michigan, a member of the National Nanotechnology Infrastructure Network funded by the National Science Foundation.

1. Catalyst Patterning for SWNT Growth
<ol>
	<li>Begin with a silicon wafer with a low pressure chemical vapor deposition (CVD) grown 500 nm Si3N4/ 500 nm SiO2 film on top.</li>
	<li>Spin coat a layer of photoresist (PR) at 500 rpm for 5 sec and then 4,000 rpm for 40 sec.</li>
	<li>Bake the wafer at 115 &deg;C for 90 sec.</li>
	<li>Use a photomask with rectangular pits for catalysts (Figure 1) and expose the wafer in UV (365 nm) irradiance of 300 mJ/cm2 for 0.3 sec. After exposure bake the wafer at 115 &deg;C for 90 sec.</li>
</ol>
Tip: Design pits of different sizes e.g. 5 micron x 5 micron, 10 micron x 5 micron etc. to account for the variability in SWNT chemical vapor deposition (CVD) growth process.
<ol start="5">
	<li>Develop the wafer in developer for 70 sec gently shaking the wafer through the process.</li>
	<li>Rinse the wafer with DI water for 2 min and then blow dry with a nitrogen (N2) gun.</li>
	<li>Load the developed wafer into e-beam evaporator chamber and deposit 0.5 nm iron (Fe) at a chamber pressure of 10-6 torr.</li>
	<li>Dice the wafer into smaller dies 1.5 cm x 1.5 cm.</li>
	<li>Remove the photoresist by dipping in warm acetone and isopropanol (IPA) for 10 min each. This leaves behind Fe catalyst in the rectangular pits for nanotube growth.</li>
</ol>
2. CVD Growth of Carbon Nanotubes
<ol>
	<li>Place the catalyst coated dies in the quartz tube of the CVD growth furnace.</li>
</ol>
Tip: Determine sweet spot for nanotube growth. Growth is uniform over an area of 2inch x 2inch downstream for our furnace (Figure 2c).
<ol start="2">
	<li>Anneal the substrate in air at 880 &deg;C for one hour to remove photoresist residue (Figure 2a). Allow to cool down.</li>
	<li>Purge the chamber with Argon for 5 min at 3 SLM (standard liter per minute).</li>
	<li>Ramp up the furnace to 800 &deg;C at the center of the tube, while maintaining a flow of 1 SLM of argon (Figure 2b).</li>
	<li>Flow 0.2 SLM of hydrogen for 5 min to reduce the catalyst particles i.e. convert iron oxide to iron.</li>
	<li>Introduce 5.5 sccm (standard cubic centimeter per minute) of ethylene (C2H4) for 35 min to grow SWNTs. Maintain a H2 flow of 0.2 SLM throughout the process. The length of SWNTs obtained from this recipe is &gt; 20 micron (&mu;m).</li>
	<li>Allow the furnace to cool down to room temperature with a small argon flow.</li>
</ol>
3. SWNT FET Transistor Fabrication
<ol>
	<li>Design a photomask (Figure 1) for defining source-drain electrodes for current-voltage (I-V) characterization of carbon nanotubes.</li>
</ol>
Tip: Extend electrode contact pads far apart on the die so that they remain accessible even after putting down a micro-fluidic stamp on the active nanotube region.
<ol start="2">
	<li>Follow steps 1.2 -1.6 to define area for metal deposition for contacts.</li>
	<li>Deposit Ti/Au 0.5 nm/50 nm for source-drain contacts in an e-beam evaporator chamber at 10-6 torr.</li>
	<li>Leave the die in acetone overnight for metal liftoff. After liftoff, dip the die in IPA for 10 min and then blow dry using N2 gun.</li>
	<li>Do a blanket deposition of 500 nm e-beam evaporated SiO2 for gate dielectric at 10-6 torr.</li>
	<li>Design a photomask (Figure 1) for defining the gate electrode.</li>
	<li>Follow steps 1.2-1.6 to define region for gate metal deposition.</li>
	<li>Evaporate 50 nm/50 nm Cr/Au as the top gate electrode in e-beam evaporator. Follow step 3.4 for metal liftoff.</li>
</ol>
Tip: Use thick chrome layer to increase strength of suspended top gate. Gate dimensions are also critical for successful suspension.
<ol start="9">
	<li>Blanket deposit a thin layer (20 nm) of SiO2 for top electrode passivation.</li>
	<li>Design a photomask (Figure 1) to open a trench in SiO2 to access the carbon nanotube channel. Design pad-etch area in the same mask to open up region for accessing source, drain and gate electrodes far away from the SWNT channel.</li>
	<li>Follow steps 1.2-1.6 to pattern the PR to open up the trench for wet etch of SiO2.</li>
	<li>Wet etch the evaporated SiO2 using 1:20 BHF solution for 3 min and 30 sec. Si3N4 provides a etch stop layer to prevent further penetration of BHF.</li>
</ol>
Tip: Etch time calibration is recommended.
<ol start="13">
	<li>Thoroughly rinse the device in DI water, and then dip it in IPA for 5 min. Blow dry using a N2 gun. The device structure remains intact due to the thick chrome layer.</li>
</ol>
4. Chemical Functionalization of Carbon Nanotube Sidewalls
<ol>
	<li>Prepare a solution of 6 mM 1-pyrenebutanoic acid succinimidyl ester (PBSE) in dimethylformamide (DMF).</li>
	<li>Incubate the SWNT FET die in this linker molecular solution for 1 hr at room temperature.</li>
	<li>Thoroughly rinse the die in DMF to wash away the excess reagent. Blow-dry the device.</li>
	<li>Prepare a 20mg/ml solution of biotinyl-3, 6-dioxaoctanediamine (biotin PEO amine -BPA) in DI water for biotinylation of SWNTs.</li>
	<li>Incubate the die in this solution for 18 hr after which thoroughly rinse the die in DI water and blow dry. BPA attaches to the PBSE linker molecule.</li>
	<li>Prepare a solution of 1mg/ml streptavidin in 7.2 pH PBS solution for streptavidin binding.</li>
	<li>For static measurements, incubate the die in streptavidin solution for 20 min to completely functionalize the biotinylated SWNT. Thoroughly rinse and blow dry the die. For real time sensing, stamp the PDMS flow channel (step 6) first and then flow the streptavidin solution in high ionic strength background for streptavidin binding (Figure 3).</li>
</ol>
Note: We rinse the die by dispensing DI water (~50 ml) over the die using a squeeze bottle. Then we move the die to another Petri dish containing DI water and move the die around for 1 min. We repeat the two steps a total of 8-10 times.
5. Preparation of Polydimethylsiloxane (PDMS) Mold for Fluid Chamber
<ol>
	<li>In a weighing cup, pour 9 parts by weight of PDMS monomer and add 1 part by weight of curing agent and thoroughly mix the two.</li>
	<li>Degas the mixture in a desiccator for 25 min. The bubbles will rise through the mixture and leave.</li>
</ol>
Tip: If the mixture starts foaming, vent the chamber and let it settle down for a few seconds before degassing again.
<ol start="3">
	<li>Place a new silicon wafer in a Petri dish. Pour the degassed PDMS mixture on top of it to have a PDMS layer of 5 mm above the wafer.</li>
	<li>Place the Petri dish in an oven at 70 &deg;C for 1 hr.</li>
	<li>Remove the Petri dish and let it cool down. Use a scalpel to cut out a rectangular piece of PDMS and pull it out using a tweezer.</li>
</ol>
Tip: The PDMS side directly in contact with the silicon wafer is clean and extremely flat. This side will be in contact with the SWNT FET die. Be careful not to contaminate it.
<ol start="6">
	<li>Place the rectangular die upside down and drill a hole in it using a biopsy punch (3 mm diameter) from the flat side to the other. This ensures no rough edges on the flat side of PDMS (Figure 4a).</li>
	<li>Place the PDMS chamber on top of the device carefully by aligning it on top of the active area of the fabricated SWNT FET dies (Figure 4a, b). Tap it gently to secure the stamp on die. The flat side bonds to the die to provide a leak-tight chamber.</li>
</ol>
Tip: This can be done with the naked eye or using an optical microscope with enough working space. If the PDMS does not stick well (generally if the die and/or the PDMS stamp is not clean), do oxygen plasma (20 watts, 15 sec) on PDMS to assist bonding. Using plasma powers higher than this leads to stronger bonding, however, we have seen ripping of electrodes while removing the PDMS in such case.
<ol start="8">
	<li>Remove the PDMS stamp after electrical testing and before next chemical functionalization step by dipping the die in DI water and gently lifting the stamp.</li>
</ol>
6. Preparation of Microfluidic Flow Channel
<ol>
	<li>Take a clean silicon wafer and place it on hot plate at 200 &deg;C for 5 min to remove any moisture.</li>
	<li>Spin coat SU-8 2015 at 500 rpm (100 rpm/sec ramp rate) for 5 sec and then 1,250 rpm for 30 sec at 300 rpm/sec ramp rate. This gives a 30 &mu;m thick SU-8 layer on silicon wafer.</li>
	<li>Soft bake the wafer at 95 &deg;C for 5 min.</li>
	<li>Design a photomask (Figure 4c) to define the 300 &mu;m wide flow channel pattern on top of the SWNT area.</li>
</ol>
Tip: To avoid collapse of structure a channel width: height ratio of 10:1 is sufficient (300 &mu;m: 30 &mu;m in this case).
<ol start="5">
	<li>Using 365-nm UV photolithography define the flow channel with a UV exposure time of 0.9 sec.</li>
	<li>Post bake the die at 95 &deg;C for 5 min.</li>
	<li>Develop the pattern in SU-8 developer for 5 min accompanied by gentle shaking.</li>
	<li>Rinse the wafer in IPA and blow dry with N2 gun.</li>
	<li>Follow steps 5.1-5.2 to prepare a PDMS mixture.</li>
	<li>Place the wafer with SU-8 mold in a desiccator along with a 2-3 drop of silanizing agent trichloro(3, 3, 3-trifluoropropyl)silane in a Petri dish. Turn on the vacuum pump let the wafer sit in vacuum for 1 hr.</li>
	<li>Pour the degassed PDMS mixture onto the wafer and heat it in an oven at 70 &deg;C for 1 hr.</li>
	<li>Cut the PDMS mold (negative of SU-8) using a scalpel.</li>
	<li>Place the PDMS stamp upside down and using a biopsy punch (0.75 mm diameter) to drill a hole at each end of the flow channel. Make sure to drill the hole from the flat side (the side in contact with the silicon wafer) to the other to avoid rough edges (Figure 4e).</li>
	<li>Place the PDMS flow chamber on top of the device carefully by aligning it on top of the active area of the fabricated SWNT FET dies under a microscope. Tap it gently to secure the stamp on die. The flat side bonds to the die to provide a leak-tight chamber. (Figure 4c and 4d)</li>
	<li>Push a polyethylene tube into the holes and connect the other end to a fluid source and drain syringe (Figure 4e).</li>
	<li>Attach the syringe to a syringe pump system to maintain a controlled flow of the fluid through the channel (Figure 6).</li>
</ol>
7. DC Electrical Measurement Setup
<ol>
	<li>Connect the source and gate contacts of SWNT FET to voltage ports of a DAQ card.</li>
	<li>Connect the drain contact to input port of DAQ card through a current pre-amplifier.</li>
	<li>Apply 30 millivolt (mV) to source contact, sweep the gate voltage and record the current from the drain (Figure 5a).</li>
</ol>
Tip: For measurements in solution, keep gate voltage sweep parameter within |0.7 volts| to avoid leakage and reaction between the gate metal electrode and solution.
8. AC Electrical Measurement Setup
<ol>
	<li>Connect the Ref-out signal from lock-in amplifier to the external modulation signal port on frequency generator to setup AM modulated frequency output.</li>
	<li>Connect source contact of SWNT FET to the AM-modulated RF output port of frequency generator and dc voltage from DAQ card using a bias tee (Figure 5b and 5c).</li>
	<li>Connect gate contact to voltage port of DAQ card.</li>
	<li>Connect the drain contact to a lock-in amplifier to read the ac current through the nanotube. Read the amplitude and phase of current through the DAQ input ports.</li>
	<li>Hold source dc voltage at 0 volt and AM signal frequency at 200 kilohertz (kHz).</li>
	<li>Sweep the gate voltage and measure the current from the drain.</li>
	<li>Increase frequency and repeat step 8.6 for Imix-Vg sweeps at different frequencies.</li>
</ol>
9. Electrical Measurements in Solution (No Flow)
<ol>
	<li>Prepare 1 mM NaCl, 10 mM NaCl and 100 mM NaCl salt solutions starting from 5M NaCl stock solution.</li>
	<li>Take the SWNT device from step 3.13. Carry out steps 4.1-4.5 to obtain a biotinylated device.</li>
	<li>Follow step 5 to put a PDMS chamber on top of the device.</li>
	<li>Fill the chamber with DI water using a pipette.</li>
	<li>Follow step 8 for frequency mixing measurements for different frequencies.</li>
	<li>Repeat 9.4 for the three different salt solutions 1 mM NaCl, 10 mM NaCl and 100 mM NaCl.</li>
</ol>
Tip: Use pipette to withdraw previous solution and then flush the chamber multiple times with the new solution. Always switch from the low to high concentration solutions.
<ol start="7">
	<li>Remove the PDMS stamp by gently lifting the stamp with a tweezer.</li>
	<li>Thoroughly rinse the device with DI water.</li>
	<li>Carry out streptavidin binding as explained in 4.6-4.7.</li>
	<li>Repeat steps 9.3-9.6.</li>
</ol>
10. Electrical Measurement in Solution (Real Time Flow)
<ol>
	<li>Prepare 100 mM NaCl salt solution starting from 5 M NaCl stock solution.</li>
	<li>Take the SWNT device from step 3.13. Carry out steps 4.1-4.5 to obtain a biotinylated device.</li>
	<li>Place the micro-fluidic flow channel on the device following step 6.. Connect an empty syringe to one end of the micro-fluidic channel for withdrawal mode operation. At the other end attach a syringe with background solution of 100 mM NaCl.</li>
	<li>Set up the electrical measurement as detailed in step 8. Fix the frequency (f = 10 MHz) and gate voltage bias (V = 0).</li>
	<li>Start syringe pump in withdrawal mode (flow rate = 0.4 ml hour-1) and monitor the current signal with time. Switch the solution to 1mg/ml streptavidin in 100 mM NaCl and monitor signal change for streptavidin-biotin binding (Figure 6).</li>
</ol>

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The authors declare that they have no competing financial interests.

The growth of carbon nanotubes depends not only on furnace conditions but also substrate cleanliness. The optimal gas flow rate, temperature and pressure for growth have to carefully calibrated and once fixed they are more or less stable. Even with these conditions being met, we found that growth depends on the patterned catalyst area, amount of catalyst and substrate cleanliness. Hence, we incorporated multiple catalyst pit sizes to account for the variability in growth. A one hour high temperature anneal step helped re...

When a charged molecule binds to a SWNT or NW electronic sensor, it can either donate/accept electrons or act as a local electrostatic gate. In either case, the bound molecule can alter the charge density in the SWNT or NW channel, leading to a change in the measured DC conductance of the sensor. A large variety of molecules15-20 have been successfully detected by studying DC characteristics of the nanosensors during such binding events. Even though charge-detection based sensing mechanism has many advantages including label-free detection21, femto-molar sensitivity22, and electronic read out capability15; it is effective only in low ionic strength solutions. In high ionic strength solutions, DC detection is impeded by ionic screening6-8. A charged surface attracts counter-ions from the solution which forms an electrical double layer (EDL) near the surface. The EDL effectively screens off these charges. As the ionic strength of the solution increases, the EDL becomes narrower and the screening increases. This screening effect is characterized by the Debye screening length &lambda;D,
<img alt="Equation 1" fo:src="/files/ftp_upload/50438/50438eq1highres.jpg" src="/files/ftp_upload/50438/50438eq1.jpg" /> 
, where &epsilon; is the dielectric permittivity of the media, kB is the Boltzmann's constant, T is the temperature, q is the electron charge, and c is the ionic strength of the electrolyte solution. For a typical 100 mM buffer solution, &lambda;D is around 1 nm and the surface potential will be completely screened at a distance of a few nm. As the result, most of nanoelectronic sensors based on SWNTs or NWs operate either in dry state20 or in low ionic strength solutions5,15,17,21-22 (c ~ 1 nM - 10 mM); otherwise the sample needs to undergo desalting steps15,23. Point-of-care diagnostic devices need to operate in physiologically relevant ionic strengths at patient site with limited sample processing capability. Hence, mitigating ionic screening effect is critical for development and implementation of POC nanoelectronic biosensors.
We mitigate the ionic screening effect by operating SWNT based nanoelectronic sensor at megahertz frequency range. The protocol provided here details the fabrication of a SWNT transistor based nanoelectronic sensing platform and high frequency mixing measurement for biomolecular detection. The single-walled carbon nanotubes are grown by chemical vapor deposition on substrates patterned with Fe catalysts24. For our SWNT transistors, we incorporate a suspended top-gate25 placed 500 nm above the nanotube, which helps enhance high frequency sensor response and also allows for a compact micro-fluidic chamber to seal the device. The SWNT transistors are operated as high frequency mixers9-11 in order to overcome the background ionic screening effects. At high frequencies, the mobile ions in solution do not have sufficient time to form the EDL and the fluctuating biomolecular dipoles can still gate the SWNT to generate a mixing current, which is our sensing signal. The frequency mixing arises due to the nonlinear I-V characteristics of a nanotube FET. Our detection technique differs from the conventional techniques of charge based detection and impedance spectroscopy26-27. Firstly, we detect biomolecular dipoles at high frequency rather than the associated charges. Secondly, the high transconductance of SWNT transistor provides an internal gain for the sensing signal. This obviates the need for external amplification as in case of high frequency impedance measurements. Recently, other groups have also addressed biomolecular detection in high background concentrations23,28. However, these methods are more involved, requiring complex fabrication or careful chemical engineering of receptor molecules. Our high frequency SWNT sensor incorporates a simpler design and utilizes the inherent frequency mixing property of a nanotube transistor. We are able to mitigate the ionic screening effects, thus promising a new biosensing platform for real-time point-of-care detection, where biosensors functioning directly in physiologically relevant condition are desired.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents which were provided within Lurie Nanofabrication Facility (University of Michigan) are marked as LNF in the catalogue column. Chemicals which require protective equipment (gloves, safety goggles, face mask, apron) and/or fume hood are denoted with PPE in comments section.</td>
		</tr>
		<tr>
			<td>Silicon wafers (P-type, &lt;100&gt;, 500-550 &mu;m thick)</td>
			<td>Silicon Valley Microelectronics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SPR 220 3.0</td>
			<td>Dow (Rohm and Haas)</td>
			<td>Megaposit SPR</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>AZ 300MIF</td>
			<td>AZ Electronic Material Corporation</td>
			<td>&nbsp;</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>J T Baker</td>
			<td>9005-05</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Isopropanol (IPA)</td>
			<td>J T Baker</td>
			<td>9079-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffered Hydrofluoric Acid</td>
			<td>Transene</td>
			<td>&nbsp;</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>1-Pyrene Butanoic Acid, succinimidyl ester</td>
			<td>Molecular Probes</td>
			<td>P130</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Biotin PEO Amine</td>
			<td>Thermo Scientific</td>
			<td>EZ- Link PEG2 Biotin, # 21346</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Invitrogen</td>
			<td>S 888</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>MP Biomedicals</td>
			<td>0219514791</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane Elastomer Base and Curing Agent</td>
			<td>Dow Corning</td>
			<td>Sylgard 184 Elastomer Kit</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>SU-8 2015</td>
			<td>Microchem</td>
			<td>Y111064</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Silanizing agent</td>
			<td>Sigma Aldrich</td>
			<td>452807</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Hydrogen</td>
			<td>Purity Plus</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethylene</td>
			<td>Purity Plus</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Argon</td>
			<td>Purity Plus</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline System</td>
			<td>Sigma Aldrich</td>
			<td>PBS1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment provided by Lurie Nanofabrication Facility (University of Michigan) is denoted as LNF in Catalogue column.</td>
		</tr>
		<tr>
			<td>GCA 200 Autostepper</td>
			<td>GCA</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Low Pressure Chemical Vapor Deposition Tool</td>
			<td>Tempress</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>e-beam Evaporator</td>
			<td>Enerjet</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CNT growth Furnace</td>
			<td>First Nano</td>
			<td>Easy Tube 3000 (LNF)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Photomasks</td>
			<td>Nanofilm</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dish (150mm)</td>
			<td>&nbsp;</td>
			<td>LNF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Bel-Art</td>
			<td>F420100000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>Ted Pella</td>
			<td>15071/78</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Ted Pella</td>
			<td>548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene Tubing PE-50</td>
			<td>VWR</td>
			<td>20903-414</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher Scientific</td>
			<td>BD Safety-Lok Syringes</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe Needles</td>
			<td>Fisher Scientific</td>
			<td>14-821-13A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAQ card</td>
			<td>National Instruments</td>
			<td>779111-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GPIB connector</td>
			<td>National Instruments</td>
			<td>778032-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lock-in Amplifier</td>
			<td>Stanford Research Systems</td>
			<td>SR 830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Frequency Generator</td>
			<td>HP Agilent</td>
			<td>8648B, 9kHz -2GHz</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bias Tee</td>
			<td>Picosecond</td>
			<td>5575A-104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Current Preamplifier</td>
			<td>DL Instruments, LLC</td>
			<td>DL 1211</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td>Allied Electronics</td>
			<td>665-xxxx</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SMA cables</td>
			<td>Sentro Tech Corp</td>
			<td>SCF65141</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

fabrication of carbon nanotube high-frequency nanoelectronic biosensor for sensing in high ionic strength solutions

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

A scanning electron microscope image of SWNT transistor with a suspended top gate is shown in Figure 7a. The gate dimensions are critical for suspension25. The current design dimensions are (length x width x thickness = 25 &mu;m x 1 &mu;m x 100 nm). The gate electrode consists of 50 nm Cr/50 nm Au; a thick chrome layer adds more strength to suspended structure. The suspended structure is confirmed by absence of leakage current between top gate and drain (Figure 7b).
<p cla...

Watch this Scientific Journal Video about Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions at JoVE.com

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

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

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

fabrication-carbon-nanotube-high-frequency-nanoelectronic-biosensor

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18.1K Views.  University of Michigan - Ann Arbor. We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

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.

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 ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...