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

Summary

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.

Abstract

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.

Introduction

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 ions^5,6, bacterial toxins⁷, dopamine⁸, DNA^9-11, RNA^12,13, enzyme and cancer biomarkers^14-19, human hormones²⁰, and cytokines^21,22. 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'-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.

Protocol

1. Fabrication and Preservation of pSNWFET Devices

1. Device Fabrication
   Note: The pSNWFET was fabricated using a sidewall spacer technique as previously reported^23,24.
   1. Prepare the gate dielectric layer.
      1. 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 °C for 4 hr).
      2. Deposit a 50-nm-thick nitride (Si₃N₄) layer by using low-pressure chemical vapor deposition (LPCVD)²⁵ at 980 °C for 4 hr.
   2. Deposit a 100-nm-thick tetraethyl orthosilicate (TEOS) layer by using LPCVD²⁵ at 780 °C for 4 hr.
   3. Perform standard lithography by using an I-line stepper to define the oxide dummy structures.
      1. Coat the wafer surface with an 830-nm-thick photoresist layer.
      2. Insert a pattern-defined photomask into the I-line stepper.
      3. Process the exposure by using the I-line stepper (wavelength: 365 nm) at a strength of 1,980 J at room temperature.
      4. Develop the wafer within a developer for 5 min.
      5. Perform the isotropic etching process by using a standard inductively coupled plasma²⁵ etcher with HBr and Cl plasma gas for 1 min.
   4. Deposit a 100-nm-thick amorphous silicon (α-Si) layer by using LPCVD²⁵.
   5. Perform an annealing step at 600 °C in N₂ ambient for 24 hr to transform the α-Si into a polycrystalline structure.
   6. Implant phosphorous ions through source/drain (S/D) doping at low energy (5E¹⁵ cm⁻²)²⁵.
   7. 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.
   8. Perform the passivation process (200-nm-thick TEOS oxide layer) by using LPCVD at 780 °C for 5 hr²⁵.
   9. 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.
      1. Repeat step 1.1.3.
      2. Perform the wet etching process (DHF: HF/H₂O for 1 min).
2. Wafer preservation
   1. Seal the wafer in a vacuum storage bag and store it in an electronic dry cabinet (relative humidity <40% at room temperature).

2. Pretreatment of the Device

1. Device cleaning
   1. Rinse the device with pure acetone.
   2. Sonicate (46 kHz, 80 W) the device in pure acetone for 10 min.
   3. Sonicate (46 kHz, 80 W) the device in pure ethanol (99.5%) for 5 min.
   4. Blow-dry the surface of the device with nitrogen.
2. Oxygen plasma
   1. Treat the device with O₂ plasma at 18 W for 30 sec.

3. Immobilization of the DNA Probe on the Device Surface

1. Amine group modification
   1. Immerse the device in a 2.0% (3-aminopropyl)triethoxysilane (APTES)/ethanol solution for 30 min.
   2. Wash the device with ethanol three times, and then sonicate (46 kHz, 80 W) the device in ethanol for 10 min.
   3. Heat the device on a hot plate at 120 °C for 10 min to create amine groups on SNWs.
2. Aldehyde group modification
   1. Immerse the device in 12.5% glutaraldehyde for 1 hr at room temperature to create aldehyde groups on the surface. Avoid light exposure.
   2. 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.
3. DNA probe immobilization
   1. Immerse the device in a solution containing 1 µM DNA probes overnight.
   2. 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.
   3. Wash the device with Na-PB (pH 7.0) three times, and then blow-dry the surface of the device with nitrogen.

4. Confirmation and Analysis of Surface Modification on pSNWFET

1. pH profile following each step of surface modification
   1. Prepare 10 mM Na-PB in pH 3.0-9.0.
      1. Prepare 10 mM sodium phosphate tribasic dodecahydrate (Na₃PO₄) in deionized water (pH 11.60).
      2. Prepare 10 mM phosphoric acid (H₃PO₄) in deionized water (pH 2.35).
      3. 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.
   2. AC conductance measurement
      Note: The measurement circuit included a small AC signal generator and an Au microwire that served as the liquid gate electrode.
      1. Determine the optimal liquid gate voltage (V_LG) 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.
         1. 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.
         2. Measure the real-time conductance of the device while sweeping V_LG from 0.80 to 1.30 V.
            1. Perform conductance measurement by using the lock-in technique²⁷ at room temperature.
            2. Convert the AC current signals into an AC voltage signal by using a low-noise current preamplifier.
            3. Collect data on conductance (G) upon increasing V_LG from 0.80 to 1.30 V (interval = 0.02 V).
         3. Determine the optimal V_LG (most sensitive V_LG) of the device²⁶.
            1. Plot the G-V_LG curves from step 4.1.2.1.2.3 to obtain the equation of the curve.
            2. Differentiate the equation, and calculate the values in each point of V_LG.
            3. Divide the value from step 4.1.2.1.3.2 by G, and determine the optimal V_LG according to the maximum number.
      2. Measure the real-time conductance of the pH profile at each step of the surface modification.
         1. Perform conductance measurement by using the lock-in technique²⁷ at room temperature.
         2. Convert the AC current signal into AC voltage signals by using a low-noise current preamplifier.
         3. Set the optimal V_LG for each step of surface modification (step 2.2: V_LG = 1.02, step 3.1: V_LG = 0.98, step 3.2: V_LG = 0.98, and step 3.3: V_LG = 1.0).
         4. 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.
2. Measurement of electric properties (the I_D-V_BG 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.
   1. 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.
   2. Measure the I_D of the device (steps 2.2, 3.1, 3.2, and 3.3) by using a commercial semiconductor analyzer and software.
      1. Select the "nMOSFET" mode.
      2. Select "I_D-V_BG" modules.
         Note: I_D is the drain/source current, and V_BG is the back gate voltage.
      3. Set a constant bias voltage (V_D = 0.5 V) while sweeping the gate potential (V_BG) from −1 to 3.0 V (interval = 0.2 V).
      4. Click the Run icon to obtain I_D-V_BG curves.

5. DNA Biosensing

Note: In a typical experiment, the I_D-V_BG curve is determined three times to ensure that no further variation is observed.

1. Determination of the baseline
   1. 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.
   2. Measure the I_D of the device (repeat step 4.2.2).
2. Sensing of DNA/DNA hybridization
   1. 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.
   2. 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.
   3. Repeat step 4.2.2 to measure the I_D of the device.
3. Reconfirm the signal of DNA/DNA hybridization.
   1. 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.
   2. 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).
   3. Repeat step 4.2.2 to measure the I_D of the device.
4. Negative control
   1. 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.
   2. 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).
   3. Repeat step 4.2.2 to measure the I_D of the device.

Results

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 ...

Discussion

Commercializing the top-down and bottom-up fabrication approaches for sSNWFETs is considered difficult because of its cost^32,33, SNW position control^34,35, 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 pre...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetone	ECHO	AH-3102
(3-Amonopropyl)triethoxysilane (APTES), ≥98%	Sigma-Aldrich	A3648	Danger
Ethanol, anhydrous, 99.5%	ECHO	484000203108A-72EC
Glutaraldehyde solution (GA), 50%	Sigma-Aldrich	G7651	Avoid light
Sodium cyanoborohydride, ≥95.0%	Fluka	71435	Danger and deliquescent
Sodium phosphate tribasic dodecahydrate, ≥98%	Sigma	04277
Phosphoric acid, ≥99.0%	Fluka	79622	Deliquescent
Photoresist (iP3650)	Tokyo Ohka Kogyo Co., LTD	THMR-iP3650 HP
Synthetic oligonucleotides, HPLC purified	Protech Technology
Tris(hydroxymethyl)aminomethane (Tris), ≥99.8%	USB	75825
Keithley 2636 System SourceMeter	Keithley
SR830 DSP Lock-In Amplifier	Stanford Research Systems
SR570 Low-noise Current Preamplifier	Stanford Research Systems
Ni PXI Express	National Instruments