This research was financially supported by Ministry of Science and Technology, Taiwan (104-2514-S-009 -001, 104-2627-M-009-001 and 102-2311-B-009-004-MY3). We thank the National Nano Device Laboratories (NDL) for its valuable assistance during device fabrication and analysis.

1. Fabrication and Preservation of pSNWFET Devices
<ol>
	<li>Device Fabrication 
	Note: The pSNWFET was fabricated using a sidewall spacer technique as previously reported23,24.
	<ol>
		<li>Prepare the gate dielectric layer.
		<ol>
			<li>Cap a 100-nm-thick thermal oxide (SiO2) layer on a Si substrate by using the wet oxidation process25 (O2 and H2 process gas at 980 &#176;C for 4 hr).</li>
			<li>Deposit a 50-nm-thick nitride (Si3N4) layer by using low-pressure chemical vapor deposition (LPCVD)25 at 980 &#176;C for 4 hr.</li>
		</ol>
		</li>
		<li>Deposit a 100-nm-thick tetraethyl orthosilicate (TEOS) layer by using LPCVD25 at 780 &#176;C for 4 hr.</li>
		<li>Perform standard lithography by using an I-line stepper to define the oxide dummy structures.
		<ol>
			<li>Coat the wafer surface with an 830-nm-thick photoresist layer.</li>
			<li>Insert a pattern-defined photomask into the I-line stepper.</li>
			<li>Process the exposure by using the I-line stepper (wavelength: 365 nm) at a strength of 1,980 J at room temperature.</li>
			<li>Develop the wafer within a developer for 5 min.</li>
			<li>Perform the isotropic etching process by using a standard inductively coupled plasma25 etcher with HBr and Cl plasma gas for 1 min.</li>
		</ol>
		</li>
		<li>Deposit a 100-nm-thick amorphous silicon (&#945;-Si) layer by using LPCVD25.</li>
		<li>Perform an annealing step at 600 &#176;C in N2 ambient for 24 hr to transform the &#945;-Si into a polycrystalline structure.</li>
		<li>Implant phosphorous ions through source/drain (S/D) doping at low energy (5E15 cm&#8722;2)25.</li>
		<li>Perform standard lithography by using the I-line stepper to remove the poly-Si layer and form the polysilicon nanowire (pSNW)25. 
		Note: Repeat step 1.1.3 to implant dopants in places other than S/D regions with poly-Si removal and form the sidewall Si channels in a self-aligned manner.</li>
		<li>Perform the passivation process (200-nm-thick TEOS oxide layer) by using LPCVD at 780 &#176;C for 5 hr25.</li>
		<li>Perform standard lithography by using the I-line stepper to expose the nanowire channels, and form test pads by using the two-step dry/wet etching process.
		<ol>
			<li>Repeat step 1.1.3.</li>
			<li>Perform the wet etching process (DHF: HF/H2O for 1 min).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Wafer preservation
	<ol>
		<li>Seal the wafer in a vacuum storage bag and store it in an electronic dry cabinet (relative humidity &#60;40% at room temperature).</li>
	</ol>
	</li>
</ol>
2. Pretreatment of the Device
<ol>
	<li>Device cleaning
	<ol>
		<li>Rinse the device with pure acetone.</li>
		<li>Sonicate (46 kHz, 80 W) the device in pure acetone for 10 min.</li>
		<li>Sonicate (46 kHz, 80 W) the device in pure ethanol (99.5%) for 5 min.</li>
		<li>Blow-dry the surface of the device with nitrogen.</li>
	</ol>
	</li>
	<li>Oxygen plasma
	<ol>
		<li>Treat the device with O2 plasma at 18 W for 30 sec.</li>
	</ol>
	</li>
</ol>
3. Immobilization of the DNA Probe on the Device Surface
<ol>
	<li>Amine group modification
	<ol>
		<li>Immerse the device in a 2.0% (3-aminopropyl)triethoxysilane (APTES)/ethanol solution for 30 min.</li>
		<li>Wash the device with ethanol three times, and then sonicate (46 kHz, 80 W) the device in ethanol for 10 min.</li>
		<li>Heat the device on a hot plate at 120 &#176;C for 10 min to create amine groups on SNWs.</li>
	</ol>
	</li>
	<li>Aldehyde group modification
	<ol>
		<li>Immerse the device in 12.5% glutaraldehyde for 1 hr at room temperature to create aldehyde groups on the surface. Avoid light exposure.</li>
		<li>Wash the device with 10 mM sodium phosphate buffer (Na-PB; pH 7.0) three times, and then blow-dry the surface of the device with nitrogen.</li>
	</ol>
	</li>
	<li>DNA probe immobilization
	<ol>
		<li>Immerse the device in a solution containing 1 &#181;M DNA probes overnight.</li>
		<li>Immerse the device in 10 mM Tris buffer (pH 8.0) with 4.0 mM NaBH3CN for 30 min to block the unreacted aldehyde groups.</li>
		<li>Wash the device with Na-PB (pH 7.0) three times, and then blow-dry the surface of the device with nitrogen.</li>
	</ol>
	</li>
</ol>
4. Confirmation and Analysis of Surface Modification on pSNWFET
<ol>
	<li>pH profile following each step of surface modification
	<ol>
		<li>Prepare 10 mM Na-PB in pH 3.0-9.0.
		<ol>
			<li>Prepare 10 mM sodium phosphate tribasic dodecahydrate (Na3PO4) in deionized water (pH 11.60).</li>
			<li>Prepare 10 mM phosphoric acid (H3PO4) in deionized water (pH 2.35).</li>
			<li>Place the pH electrode into 500 ml of 10 mM Na3PO4 buffer, and mix this solution with different volumes of 10 mM H3PO4 buffer while measuring the pH value to obtain buffers with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0.</li>
		</ol>
		</li>
		<li>AC conductance measurement 
		Note: The measurement circuit included a small AC signal generator and an Au microwire that served as the liquid gate electrode.
		<ol>
			<li>Determine the optimal liquid gate voltage (VLG) for measurement26 at each modification step (steps 2.2, 3.1, 3.2, and 3.3). 
			Note: The electric properties of the device after four surface modifications on the SNW were measured as described in this section. In step 2.2, the unmodified pSNWFET containing the native oxide layer is modified; step 3.1 entails modifying the device with the amine group of APTES; step 3.2 involves modification with the uncharged functional group of glutaraldehyde; and step 3.3 involves DNA probe modification.
			<ol>
				<li>Deliver the 10 mM Na-PB (pH 7.0) solution to the SNW surface by using a syringe pump (flow rate: 5.0 ml/hr) for direct contact with the SNW.</li>
				<li>Measure the real-time conductance of the device while sweeping VLG from 0.80 to 1.30 V.
				<ol>
					<li>Perform conductance measurement by using the lock-in technique27 at room temperature.</li>
					<li>Convert the AC current signals into an AC voltage signal by using a low-noise current preamplifier.</li>
					<li>Collect data on conductance (G) upon increasing VLG from 0.80 to 1.30 V (interval = 0.02 V).</li>
				</ol>
				</li>
				<li>Determine the optimal VLG (most sensitive VLG) of the device26.
				<ol>
					<li>Plot the G-VLG curves from step 4.1.2.1.2.3 to obtain the equation of the curve.</li>
					<li>Differentiate the equation, and calculate the values in each point of VLG.</li>
					<li>Divide the value from step 4.1.2.1.3.2 by G, and determine the optimal VLG according to the maximum number.</li>
				</ol>
				</li>
			</ol>
			</li>
			<li>Measure the real-time conductance of the pH profile at each step of the surface modification.
			<ol>
				<li>Perform conductance measurement by using the lock-in technique27 at room temperature.</li>
				<li>Convert the AC current signal into AC voltage signals by using a low-noise current preamplifier.</li>
				<li>Set the optimal VLG for each step of surface modification (step 2.2: VLG = 1.02, step 3.1: VLG = 0.98, step 3.2: VLG = 0.98, and step 3.3: VLG = 1.0).</li>
				<li>Deliver the 10 mM Na-PB solution with pH values from 3.0 to 9.0 to the SNW surface (flow rate: 5.0 ml/hr), and collect the data on conductance at a drain voltage of 0.01 V.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measurement of electric properties (the ID-VBG curve) of the device in 10 mM Na-PB (pH 7.0) following each step of surface modification (steps 2.2, 3.1, 3.2, and 3.3.) 
	Note: The electric properties of the device after four surface modifications on the SNW were measured: in step 2.2, the unmodified pSNWFET containing the native oxide layer is modified; step 3.1 involves modifying the device with the amine group of APTES; step 3.2 entails modification with the uncharged functional group of glutaraldehyde; and step 3.3 entails DNA probe modification.
	<ol>
		<li>Deliver the 10 mM Na-PB (pH 7.0) solution to the SNW surface (steps 2.2, 3.1, 3.2, and 3.3) by using a syringe pump (flow rate: 5.0 ml/hr) for direct contact with the SNW.</li>
		<li>Measure the ID of the device (steps 2.2, 3.1, 3.2, and 3.3) by using a commercial semiconductor analyzer and software.
		<ol>
			<li>Select the &#34;nMOSFET&#34; mode.</li>
			<li>Select &#34;ID-VBG&#34; modules. 
			Note: ID is the drain/source current, and VBG is the back gate voltage.</li>
			<li>Set a constant bias voltage (VD = 0.5 V) while sweeping the gate potential (VBG) from &#8722;1 to 3.0 V (interval = 0.2 V).</li>
			<li>Click the Run icon to obtain ID-VBG curves.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. DNA Biosensing
Note: In a typical experiment, the ID-VBG curve is determined three times to ensure that no further variation is observed.
<ol>
	<li>Determination of the baseline
	<ol>
		<li>Deliver the 10 mM Na-PB solution (pH 7.0) to the DNA probe-immobilized SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr), and then incubate the SNW for 30 min.</li>
		<li>Measure the ID of the device (repeat step 4.2.2).</li>
	</ol>
	</li>
	<li>Sensing of DNA/DNA hybridization
	<ol>
		<li>Load 10 pM complementary target DNA onto the DNA probe-immobilized SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr), and then incubate the SNW for 30 min.</li>
		<li>Deliver the 10 mM Na-PB solution (pH 7.0) onto the SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr) to wash the unbound target DNA away.</li>
		<li>Repeat step 4.2.2 to measure the ID of the device.</li>
	</ol>
	</li>
	<li>Reconfirm the signal of DNA/DNA hybridization.
	<ol>
		<li>Load 1 nM recovery DNA onto the DNA probe-immobilized SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr), and then incubate the SNW for 30 min.</li>
		<li>Deliver the 10 mM Na-PB solution (pH 7.0) onto the SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr).</li>
		<li>Repeat step 4.2.2 to measure the ID of the device.</li>
	</ol>
	</li>
	<li>Negative control
	<ol>
		<li>Load 100 pM non-complementary DNA onto the DNA probe-immobilized SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr), and then incubate the SNW for 30 min.</li>
		<li>Deliver the 10 mM Na-PB solution (pH 7.0) onto the SNW surface for 10 min by using a syringe pump (flow rate: 5.0 ml/hr).</li>
		<li>Repeat step 4.2.2 to measure the ID of the device.</li>
	</ol>
	</li>
</ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well+controlled+assembly+of+silicon+nanowires+by+nanowire+transfer+method.">Well controlled assembly of silicon nanowires by nanowire transfer method.</a> Nanotechnology. 18 (445302), (2007).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+label-free+DNA+sensors+based+on+silicon+nanowires.">Sequence-specific label-free DNA sensors based on silicon nanowires.</a> Nano Lett. 4, 245-247 (2004).</li><li>McAlpine, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+nanowire+electronics+and+photonics+on+glass+and+plastic+substrates.">High-performance nanowire electronics and photonics on glass and plastic substrates.</a> Nano Lett. 3, 1531-1535 (2003).</li><li>Cui, Y., Wei, Q., Park, H., Lieber, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanowire+nanosensors+for+highly+sensitive+and+selective+detection+of+biological+and+chemical+species.">Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species.</a> Science. 293, 1289-1292 (2001).</li><li>Patolsky, F., Zheng, G., Lieber, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanowire-based+biosensors.">Nanowire-based biosensors.</a> Anal Chem. 78, 4260-4269 (2006).</li><li>Hsiao, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+poly-silicon+nanowire+field+effect+transistor+for+biosensing+application.">Novel poly-silicon nanowire field effect transistor for biosensing application.</a> Biosens Bioelectron. 24, 1223-1229 (2009).</li></ol>

The authors have nothing to disclose.

Commercializing the top-down and bottom-up fabrication approaches for sSNWFETs is considered difficult because of its cost32,33, SNW position control34,35, and its low production scale36. By contrast, fabricating pSNWFETs is simple and low cost37. Through the top-down approach and combination with the sidewall spacer formation technique (Figure 1), the size of the SNW can be controlled by adjusting the duration of reactive plasma etching. The procedures for preparing the nanowires of the pSNWFET illustrated in Figure 1a can be easily adapted to commercial semiconductor facilities. Consequently, pSNWFETs have several advantages including cost effectiveness, a simple construction technique, and a CMOS-compatible fabrication process and thus can be applied in biosensing.
In contrast to the fabrication of the semiconductor device, a process well defined in commercial facilities, the immobilization of the bioprobe on the device may vary for each specific application. The immobilization of the DNA probe on the SNW surface is illustrated in Figure 2a as an example. Other bioprobes, such as antibodies, aptamers, and enzymes, can also be immobilized on the SNW surface for detecting different targets. The following steps are critical for successfully immobilizing the DNA probe. In our protocol, the as-fabricated pSNWFET device is cleaned and treated with oxygen plasma and then immersed in an APTES/ethanol solution to create amine groups on the SNWs. Next, the device is immersed in a glutaraldehyde solution to create aldehyde groups on the surface. These groups are later conjugated to the 5&#39;-aminomodified DNA probe. Multiple-step conjunctions between cross-linker molecules and the bioprobe were required to transform the semiconductor device to a biosensor. Thus, it is crucial to ensure that every step is successful. At each step of DNA probe immobilization, XPS was used to analyze and confirm the composition and chemistry of the surface. The atomic concentrations of carbon, oxygen, and nitrogen were determined on the basis of XPS scans of the respective peaks, as shown in Figure 2b. Upon addition of the DNA probe, the most notable changes were an increase in carbon and nitrogen concentrations. An increasing trend was observed in the carbon and nitrogen concentrations at each step of the DNA probe immobilization procedure. The oxygen concentration decreased during the procedure because the native hydroxyl group became covered through surface modification. These results revealed that the DNA probe was immobilized, which is consistent with the change in electric properties, as shown in Figure 2c, d, and e. The aforementioned procedures are useful for troubleshooting in case of unsuccessful surface modification. However, they are usually performed on a separate wafer coated with a material similar to that on the nanowire surface. Direct evidence of changes in the electric properties of the modified device is required to confirm the outcome of surface modification on the device, as described in the following paragraphs.
One of the most frequently used methods for directly monitoring changes in the FET device surface is pH sensing, as shown in Figure 2d and d. Different surface modifications result in variation in the charge on the SNW surface, greatly affecting the surface potential of the pSNWFET under different environments. We used broad-range pH buffer solutions to detect changes in conductance corresponding to the functional groups on the SNW surface. From a mechanistic perspective, the increase in conductance with changing pH (increase in hydrogen ions) is consistent with the increase in the positive surface charge, which &#34;turns on&#34; the n-type FET through the accumulation of electrons. The real-time electric responses to the conductance of the pSNWFET can be measured in buffer solutions with pH values ranging from 3.0 to 9.0. The methods described in this report are useful for examining whether a semiconductor-based sensor is prepared for biosensing application.
Figure 2e illustrates a convenient method for determining the outcome of the surface modification through direct measurement of the electric properties of the device. The changes in ID-VG curves shown in Figure 2e are consistent with each stage of DNA probe immobilization on the SNW surface. According to the results, pSNWFETs can be directly used to confirm the procedure at each step of bioprobe immobilization. This result also indicates that the modified pSNWFET is prepared for biosensing application. These procedures are particularly useful when the immobilization procedures are well established. The function of a pSNWFET-based biosensor was examined by detecting AIV subtype H1 DNA as an example (Figure 3). Using probe and target DNA is suitable for illustrating the development of a novel biosensor because of the stability of the DNA molecules and the techniques available for easily obtaining the desired DNA sequence. In addition to using noncomplementary DNA as a negative control, we demonstrated DNA hybridization on the pSNWFET with a recovery system. This is particularly useful when a new system or new device is used for biosensing experiments. Many steps are involved in the process from device fabrication to biosensing application. Each step affects the final outcome. Furthermore, false positive or false negative results are usually obtained in biosensing experiments because of the complexity of the biosensing environment. Thus, controlled experiments are critical, and the recovery system demonstrated in this study can greatly facilitate identifying unexpected factors that interfere with the experimental results.
The main limitations for currently used pSNWFET-based biosensor are the availability of the pSNWFET device and the instrumentation used for measurement. However, these two limitations can be easily overcome in the near future. Many types of SNWFETs have been reported in the literature. The pSNWFET demonstrated in this study is already being fabricated using the standard semiconductor process and can be mass produced with only minor adjustments to the commercial wafer fabrication process. The instrumentation used for measurement in this study was a standard semiconductor chip analyzer. This implies that, with a proper integrated circuit installed on the device, portable instrumentation can be designed and manufactured using current electronic technology.
In conclusion, we describe the complete procedure for DNA sensing on a pSNWFET. Because the immobilization procedure strongly affects the ability of a biosensor to effectively detect biomolecules, this protocol provides step-by-step confirmation of bioprobe immobilization and the readiness of the device for DNA biosensing applications. Similar procedures can be adapted from this report for many similar biosensing applications, for which adjustments are necessary. This report also describes protocols for confirming and troubleshooting of the immobilization of the bioprobe and device surface modifications. The demand for various biosensors is increasing. The methods described in this report are an appropriate reference for preparing and developing semiconductor-based biosensors.

Silicon nanowire field-effect transistors (SNWFETs) have the advantages of ultra-high sensitivity and direct electrical responses to environmental charge variation. In n-type SNWFETs for example, when a negatively (or positively) charged molecule approaches the silicon nanowire (SNW), the carriers in the SNW are depleted (or accumulate). Consequently, the conductivity of the SNWFET decreases (or increases)1. Therefore, any charged molecule near the SNW surface of the SNWFET device can be detected. Vital biomolecules including enzymes, proteins, nucleotides, and many molecules on the cell surface are charge carriers and can be monitored using SNWFETs. With suitable modifications, particularly immobilizing a biomolecular probe on the SNW, a SNWFET can be developed into a label-free biosensor.
Surveillance using biomarkers is critical for diagnosing diseases. As shown in Table 1, several studies have used NWFET-based biosensors for label-free, ultra-high-sensitivity, and real-time detection of various biological targets, including a single virus2, adenosine triphosphate and kinase binding3, neuronal signals4, metal ions5,6, bacterial toxins7, dopamine8, DNA9-11, RNA12,13, enzyme and cancer biomarkers14-19, human hormones20, and cytokines21,22. These studies have demonstrated that NWFET-based biosensors represent a powerful detection platform for a broad range of biological and chemical species in a solution.
In SNWFET-based biosensors, the probe immobilized on the SNW surface of the device recognizes a specific biotarget. Immobilizing a bioprobe usually involves a series of steps, and it is critical that every step is properly performed to ensure the proper functioning of the biosensor. Various techniques have been developed for analyzing the surface composition, including X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurement, atomic force microscopy (AFM), and scanning electron microscopy (SEM). Methods such as AFM and SEM provide direct evidence of bioprobe immobilization on the nanowire device, whereas methods such as XPS, ellipsometry, and contact angle measurement are dependent on parallel experiments performed on other similar materials. In this report, we describe the confirmation of each modification step by using two independent methods. XPS is used to examine the concentrations of specific atoms on polysilicon wafers, and variations in the electric properties of the device are measured directly to confirm the charge variation on the SNW surface. We employ DNA biosensing by using polycrystalline SNWFETs (pSNWFETs) as an example to illustrate this protocol. Immobilizing a DNA probe on the SNW surface involves three steps: amine group modification on the native hydroxyl surface of the SNW, aldehyde group modification, and 5&#39;-aminomodified DNA probe immobilization. At each modification step, the device can directly detect the variation in the charge of the functional group immobilized on the SNW surface, because the surface charges cause local interfacial potential changes over the gate dielectric that alter the channel current and conductance1. Charges surrounding the SNW surface can electrically modulate the electric properties of the pSNWFET device; therefore, the surface properties of the SNW play a crucial role in determining the electrical characteristics of pSNWFET devices. In the reported procedures, the immobilization of a bioprobe on the SNW surface can be directly determined and confirmed through electric measurement, and the device is prepared for biosensing applications.

<table border="0" cellpadding="0" cellspacing="0" width="862">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Acetone</td>
			<td style="width:191px;">ECHO</td>
			<td style="width:179px;">AH-3102</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">(3-Amonopropyl)triethoxysilane (APTES), &#8805;98%</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">A3648</td>
			<td>Danger</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Ethanol, anhydrous, 99.5%</td>
			<td style="width:191px;">ECHO</td>
			<td style="width:179px;">484000203108A-72EC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Glutaraldehyde solution (GA), 50%</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">G7651</td>
			<td>Avoid light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Sodium cyanoborohydride, &#8805;95.0%&#160;</td>
			<td style="width:191px;">Fluka</td>
			<td style="width:179px;">71435</td>
			<td>Danger and deliquescent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">Sodium phosphate tribasic dodecahydrate, &#8805;98%</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:179px;">04277</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Phosphoric acid, &#8805;99.0%</td>
			<td style="width:191px;">Fluka</td>
			<td style="width:179px;">79622</td>
			<td>Deliquescent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Photoresist (iP3650)</td>
			<td style="width:191px;">Tokyo Ohka Kogyo Co., LTD</td>
			<td style="width:179px;">THMR-iP3650 HP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Synthetic oligonucleotides, HPLC purified</td>
			<td style="width:191px;">Protech Technology</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">Tris(hydroxymethyl)aminomethane (Tris), &#8805;99.8%</td>
			<td style="width:191px;">USB</td>
			<td style="width:179px;">75825</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Keithley 2636 System SourceMeter</td>
			<td style="width:191px;">Keithley</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">SR830 DSP Lock-In Amplifier</td>
			<td style="width:191px;">Stanford Research Systems</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">SR570 Low-noise Current Preamplifier</td>
			<td style="width:191px;">Stanford Research Systems</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Ni PXI Express</td>
			<td style="width:191px;">National Instruments</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of silicon nanowire field-effect transistor for chemical and biosensing applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

Various SNWFETs have been reported to serve as transducers of biosensors (Table 1). Single-crystalline SNWFETs (sSNWFETs) and pSNWFETs show comparable electric properties as transducers in aqueous solutions, and both have been reported to have many biosensing applications. An advantageous feature of the pSNWFET device used in this study is its simple and low-cost fabrication procedure. Figure 1a shows the key steps involved in fabricating the pSNWFET. An optical image of a die from the 6-inch wafer (Figure 1b) and SEM images (Figure 1c) of a single device (two SNWs, approximately 100 nm in width and 1.6 &#956;m in length) were obtained.
Figure 2a illustrates the procedure of DNA probe immobilization on the SNW surface. Each modification step was confirmed using XPS (Figure 2b). The pH profiles for real-time conductance (Figure 2c, d) are shown at various stages of SNW surface modification. For the unmodified pSNWFET containing the native oxide layer on the SNW surface, the hydroxyl groups (-OH) were ionized to form charged groups (-O&#8722;) with increasing pH values (black line). The decrease in conductance at pH 6.0 to 9.0 indicates that the acid dissociation constant (pKa) of the hydroxyl group can be set to approximately 7.0. Conductance likely increases at pH 3.0 to 6.0 because of the increase in the ionic strength, affecting device characteristics1. After the APTES modification, the response to the conductance of the APTES-modified device showed high variation (red line). The amine group (pKa = 4.0) of APTES can be protonated at a low pH to produce a positive charge28. Thus, the SNW conductance decreased with discrete changes in pH values from 3.0 to 9.0. After the SNW was modified with glutaraldehyde, the response was relatively stable for conductance at pH ranging from 3.0 to 9.0 (blue line). This may be attributed to the uncharged functional group that is insensitive to the change in pH environments. Finally, after the negatively charged DNA probe was immobilized, conductance slightly decreased with an increase in pH values (green line).
The shift in the electric properties of the device (Figure 2e) with different modifications confirms the change in the SNW surface. In such experiments, the ID-VG curve of the unmodified pSNWFET was used as the baseline (black line). After the SNW was immersed in APTES, the ID-VG curve of the device shifted to the left (increased current) because of the positive charge on the SNW surface caused by the amino group on APTES (black to red line). After conjugation of glutaraldehyde to the APTES-modified device, the ID-VG curve shifted back to the right because of imide bond formation. The current decreased because the positively charged amine was converted to neutrally charged imide (red to blue line). Finally, the 5&#39;-amnimodified DNA probe was introduced to bind to the APTES-glutaraldehyde-modified device. The sugar-phosphate backbone of DNA caused the ID-VG curve to shift to the far right (blue to green line), which is consistent with the effect of negative ions on n-type FET.
The detection of DNA/DNA hybridization on the pSNWFET is shown in Figure 3. The probe, target, recovery, and noncomplementary DNA sequences designed for detecting the avian influenza virus (AIV)29,30 are shown in Table 2. The ID-VG curve of the DNA probe-modified pSNWFET obtained in 10 mM Na-PB (pH 7.0) was used as the baseline (black). Subsequently, 10 pM target DNA was introduced to hybridize with the immobilized DNA probe on the SNW surface, and a clear decrease in the drain current of the device was observed (red line). The decreased ID in the n-type SNWFET implied an increased negative charge (caused by the phosphate backbone) on the SNW surface. Recovery DNA was designed to rehybridize with the target DNA and free the DNA probe31. If the recovery DNA is properly designed, the reaction thermodynamically favors the rehybridization of the target-recovery DNA duplex, because more complementary nucleotides are available between these two DNA strands (Table 1) than those between the probe and target DNA. Adding 1 nM recovery DNA (blue line) further confirmed that the electric response of the target DNA was due to the hybridization of the probe-target DNA and freed the DNA probe, which is reusable for subsequent experiments. As a negative control, noncomplementary DNA [AIV subtype H5 target DNA] (100 pM) was also injected and mixed with the DNA probe, and the ID-VG curve remained unchanged. The DNA probe immobilized on the pSNWFET is insensitive to noncomplementary DNA. Only target DNA causes an appreciable shift in the ID-VG curve. The ID-VG curve returns to the baseline following incubation with the recovery DNA.
<img alt="Figure 1" src="/files/ftp_upload/53660/53660fig1.jpg" /> 
Figure 1. Preparation of the pSNWFET device. (a) Scheme of device fabrication. (i) A 6-inch Si wafer was capped with a 100-nm-thick thermal oxide layer. Next, 50-nm-thick nitride and 100-nm-thick TEOS layers were deposited as starting materials by using LPCVD. (ii) A TEOS dummy structure was defined and formed using standard lithography; two insulator layers (oxide and nitride) served as the gate dielectric. (iii) A 100-n-thick &#945;-Si layer was deposited using LPCVD, and annealing was subsequently conducted to transform the &#945;-Si into poly-Si. (iv) S/D doping was then performed through phosphorus ion implantation. (v) The sidewall Si channels were formed in a self-aligned manner by using standard lithography. (vi) Top view of the fabricated device structure with pSNW. (b) Optical images of a pSNWFET biosensor chip. (c) SEM image of a single pSNWFET device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53660/53660fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53660/53660fig2.jpg" /> 
Figure 2. Surface modification and validation on pSNWFET. (a) Scheme of surface functionalization of DNA and DNA/DNA hybridization. (b) XPS spectra of C1s, O1s, and N1s signals from the silicon wafer (sampling depth = 7.5 nm), where the sequential stepwise surface modification of the immobilized DNA probe was performed. High-resolution spectra were obtained, and the overall energy resolution was set to 0.1 eV. (c) Real-time curve at various pH values at each step of the surface modification; 10 mM Na-PB was injected in the following order: pH 7.0 &#8594; pH 6.0 &#8594; pH 5.0 &#8594; pH 4.0 &#8594; pH 3.0 &#8594; pH 4.0 &#8594; pH 5.0 &#8594; pH 6.0 &#8594; pH 7.0 &#8594; pH 8.0 &#8594; pH 9.0 &#8594; pH 8.0 &#8594; pH 7.0. (d) Average conductance at pH values from 3.0 to 9.0 with 10 mM Na-PB following each step of surface modification. (e) Electric properties (ID-VBG curves) of the pSNWFET at each step of surface modification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53660/53660fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53660/53660fig3.jpg" /> 
Figure 3. DNA biosensing on pSNWFET. The ID-VG curves of the DNA probe-modified pSNWFET were obtained in 10 mM Na-PB (pH 7) (black line). The ID-VG curve following hybridization of the probe and target DNA (red line) was obtained by introducing 10 pM target DNA and washing it with 10 mM Na-PB (pH 7). The DNA probe (blue line) was recovered by adding 1 nM recovery DNA to remove the target DNA. Noncomplementary DNA was used as negative control in this experiment (green line).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bio-target</td>
			<td>Sensitivity</td>
			<td>Crystalline</td>
			<td>Type</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>pH&#160;</td>
			<td></td>
			<td>single</td>
			<td>p</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Cystic fibrosis &#8710;F508</td>
			<td>3 bases deletion</td>
			<td>single</td>
			<td>p</td>
			<td>9</td>
		</tr>
		<tr>
			<td>Influenza A</td>
			<td>single virus</td>
			<td>single</td>
			<td>p</td>
			<td>2</td>
		</tr>
		<tr>
			<td>ATP sensing</td>
			<td>100 pM</td>
			<td>single</td>
			<td>p</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Telomerase</td>
			<td>10 Hela cell</td>
			<td>single</td>
			<td>p</td>
			<td>14</td>
		</tr>
		<tr>
			<td>PSA / CEA / Mucin-1</td>
			<td>&#60;1.4 / 2 / 5 pg/ml</td>
			<td>single</td>
			<td>n/p</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Neuronal signal</td>
			<td></td>
			<td>single</td>
			<td>n/p</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Ca2+</td>
			<td>100 nM</td>
			<td>single</td>
			<td>n</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Bacterial toxin (SEB)</td>
			<td>10 fM</td>
			<td>poly</td>
			<td>p</td>
			<td>7</td>
		</tr>
		<tr>
			<td>Dopamine</td>
			<td>1 fM</td>
			<td>poly</td>
			<td>n</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Troponin-T</td>
			<td>1 fg/ml</td>
			<td>single</td>
			<td>n</td>
			<td>19</td>
		</tr>
		<tr>
			<td>Vascular endothelial growth factor</td>
			<td>1.04 / 0.104 nM</td>
			<td>single</td>
			<td>n/p</td>
			<td>18</td>
		</tr>
		<tr>
			<td>BRAFV599E</td>
			<td>1-base-mismatch</td>
			<td>single</td>
			<td>n</td>
			<td>11</td>
		</tr>
		<tr>
			<td>AIV DNA</td>
			<td>1 fM</td>
			<td>poly</td>
			<td>n</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Avidin / streptavidin</td>
			<td>1.48 nM / 167 fM</td>
			<td>poly</td>
			<td>n</td>
			<td>37</td>
		</tr>
		<tr>
			<td>Ca2+ / Troponin I</td>
			<td>1 &#956;M / 7 nM</td>
			<td>single</td>
			<td>p</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Dengue serotype 2&#160;RNA</td>
			<td>&#60;10&#160;fM&#160;</td>
			<td>single</td>
			<td>n</td>
			<td>12</td>
		</tr>
		<tr>
			<td>CaM protein Kinase</td>
			<td>expressed cell lysate</td>
			<td>single</td>
			<td>p</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Matrix metalloproteinase-2</td>
			<td>100 fM</td>
			<td>single</td>
			<td>p</td>
			<td>15</td>
		</tr>
		<tr>
			<td>MicroRNAs (miR-21 / miR-205)</td>
			<td>1 zeptomole (ca. 600 copies)</td>
			<td>single</td>
			<td>p</td>
			<td>13</td>
		</tr>
		<tr>
			<td>Vascular endothelial growth factor</td>
			<td>1.25 pg/ml&#160;</td>
			<td>poly</td>
			<td>n</td>
			<td>21</td>
		</tr>
		<tr>
			<td>Human thyroid stimulating hormone&#160;</td>
			<td>0.11 pM&#160;</td>
			<td>single</td>
			<td>n</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Apolipoprotein A II protein</td>
			<td>6.7 pg/ml</td>
			<td>poly</td>
			<td>n</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Interleukin 8 / tumor necrosis factor &#945;&#160;</td>
			<td>10 fg/ml</td>
			<td>single</td>
			<td>n</td>
			<td>22</td>
		</tr>
	</tbody>
</table>
Table 1. Partial list of biotargets examined using SNWFET devices.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Oligonucleotides</td>
			<td>Sequence</td>
		</tr>
		<tr>
			<td>AIV H1 5&#8217;-aminomodified probe DNA</td>
			<td>5&#8217;-NH2-C6-CACACTCTGTCAACCTAC-3&#8217;</td>
		</tr>
		<tr>
			<td>AIV H1 target DNA</td>
			<td>5&#8217;-CCATTGTGACTGTCCTCAAGTAGGTTGACAGAGTGTG-3&#8217;</td>
		</tr>
		<tr>
			<td>AIV H1 recovery DNA</td>
			<td>5&#8217;-CACACTCTGTCAACCTACTTGAGGACAGTCACAATGG-3&#8217;</td>
		</tr>
		<tr>
			<td>Non-complementary DNA (AIV H5 target DNA)</td>
			<td>5&#8217;-TGATAACCAATGCAGATTTG-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 2. Sequences of synthetic oligonucleotides.

Watch this Scientific Journal Video about Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications at JoVE.com

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

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11.0K Views.  National Chiao Tung University. We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Video: Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...