Connection of ssDNA to Silicon Substrate Based on a Mechano–Chemical Method

At a Glance

Metadata	Details
Publication Date	2023-05-28
Journal	Micromachines
Authors	Liqiu Shi, Feng Yu, Mingming Ding, Zhouming Hang, Yan Feng
Institutions	Zhejiang University of Water Resource and Electric Power
Analysis	Full AI Review Included

Executive Summary

This research proposes a novel mechano-chemical method for the covalent immobilization of single-stranded DNA (ssDNA) onto a single crystal silicon substrate. This technique is critical for developing high-density DNA chips and biosensors.

Core Innovation: Covalent Si-C bonding is achieved by mechanically scribing the Si substrate using a diamond tip in a diazonium solution (benzoic acid diazo salt), generating Si free radicals that react immediately.
Coupling Mechanism: An aromatic hydrocarbon self-assembled monolayer (SAM) ending in a carboxyl group (-COOH) is formed simultaneously during scribing. ssDNA (amino-modified, -NH₂) is subsequently attached via stable amide bonds using EDC coupling.
Structural Control: The method allows for the creation of three-dimensional, controllable structures (e.g., cross-patterns, lines down to 1 µm width) on the silicon surface, enabling high-density biomolecule fixation.
Verification: XPS analysis confirmed the successful replacement of Si-O bonds with stable Si-C bonds, validating the covalent attachment of the SAM layer.
Performance Optimization: Fluorescence studies showed that the optimal ssDNA concentration for fixation and hybridization was 15 µmol/L; higher concentrations (20 µmol/L) led to decreased hybridization due to electrostatic repulsion and spatial constraints.
Significance: This strategy provides a stable, controllable platform for DNA probe fixation, laying the foundation for next-generation nano-devices and highly sensitive biosensors.

Technical Specifications

Parameter	Value	Unit	Context
Substrate Material	P-type Si(100)	N/A	Single crystal silicon wafer
Substrate Thickness	460 ± 15	µm	Used for experiments
Scribing Tool Speed	500	nm/s	Diamond tip movement rate
Diazonium Solution Conc.	50	mmol/L	Boron tetrafluoride benzoate diazo salt
SAM Assembly Time	12	hours	Required reaction time, kept dark
Roughness (Ra) Before SAM	6.511	nm	Measured via AFM
Roughness (Ra) After SAM	3.728	nm	Measured via AFM (smoother cluster shape)
Si-C/Si-O Ratio (Before)	~1:4	N/A	XPS Si2p peak area ratio
Si-C/Si-O Ratio (After)	~1:3	N/A	XPS Si2p peak area ratio (increased Si-C)
Optimal ssDNA Conc.	15	µmol/L	Maximum fluorescence brightness/hybridization
ssDNA Probe Length	24	bp	Base pairs
ssDNA Modifications	5’ FAM, 3’ NH₂	N/A	Fluorescence label (FAM) and amino coupling group (NH₂)
Characteristic FT-IR Peak	1680-1620	cm^-1	Vibration absorption peak of aryl group and -COOH connection

Key Methodologies

The process involves three primary steps: mechano-chemical functionalization of the silicon surface, formation of the aromatic coupling layer, and covalent attachment of the ssDNA probe.

Mechano-Chemical Substrate Functionalization:
- P-type Si(100) substrate is fixed in a tank containing 50 mmol/L boron tetrafluoride benzoate diazo salt solution.
- A diamond tool is moved across the surface at 500 nm/s, mechanically scribing the silicon.
- Scribing generates silicon free radicals, which immediately react covalently with the diazonium molecules in the solution, forming Si-C bonds.
Aromatic Hydrocarbon Coupling Layer (SAM) Formation:
- The covalent reaction results in a self-assembled film (SAM) on the scribed area, with the terminal group being the carboxyl group (-COOH).
- The sample is kept in the assembly solution, away from light, for approximately 12 hours to ensure sufficient reaction time.
- The sample is then rinsed thoroughly with nitrile, acetone, absolute ethanol, and ultra-pure water.
ssDNA Covalent Connection:
- The ssDNA probe is synthesized with a 3’ amino group (-NH₂) and a 5’ FAM fluorescence label.
- The substrate is immersed in a solution containing the ssDNA probe and the covalent coupling activator, N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
- The EDC facilitates a condensation reaction between the -NH₂ group of the ssDNA and the -COOH group of the SAM, forming a stable amide bond and anchoring the ssDNA to the silicon surface.
- The influence of ssDNA concentration is studied, confirming 15 µmol/L as the optimal concentration for fixation density.

Commercial Applications

The ability to create highly controlled, high-density, and stable covalent linkages of DNA probes on silicon substrates is crucial for several advanced engineering and biomedical fields.

Biosensors and Diagnostics:
- Development of highly sensitive, label-free DNA detection biosensors.
- Manufacturing of high-density DNA chips for rapid disease diagnosis and genetic sequencing.
Nano-Devices and Microelectronics:
- Integration of biomolecules into silicon-based micro- and nano-electronic devices.
- Creation of functionalized surfaces for molecular electronics and controlled surface chemistry.
Environmental Monitoring:
- Development of sensors for detecting specific microorganisms or pollutants in water systems.
- Specific application mentioned: Detecting microorganisms corroding flood gates.
Materials Science:
- Creating stable, functionalized silicon surfaces for research into surface chemistry and biomolecular immobilization techniques.

View Original Abstract

A novel fabrication process to connect single-stranded DNA (ssDNA)to a silicon substrate based on a mechano-chemical method is proposed. In this method, the single crystal silicon substrate was mechanically scribed in a diazonium solution of benzoic acid using a diamond tip which formed silicon free radicals. These combined covalently with organic molecules of diazonium benzoic acid contained in the solution to form self-assembled films (SAMs). The SAMs were characterized and analyzed by AFM, X-ray photoelectron spectroscopy and infrared spectroscopy. The results showed that the self-assembled films were covalently connected to the silicon substrate by Si-C. In this way, a nano-level benzoic acid coupling layer was self-assembled on the scribed area of the silicon substrate. The ssDNA was further covalently connected to the silicon surface by the coupling layer. Fluorescence microscopy showed that ssDNA had been connected, and the influence of ssDNA concentration on the fixation effect was studied. The fluorescence brightness gradually increased with the gradual increase in ssDNA concentration from 5 μmol/L to 15 μmol/L, indicating that the fixed amount of ssDNA increased. However, when the concentration of ssDNA increased from 15 μmol/L to 20 μmol/L, the detected fluorescence brightness decreased, indicating that the hybridization amount decreased. The reason may be related to the spatial arrangement of DNA and the electrostatic repulsion between DNA molecules. It was also found that ssDNA junctions on the silicon surface were not very uniform, which was related to many factors, such as the inhomogeneity of the self-assembled coupling layer, the multi-step experimental operation and the pH value of the fixation solution.

Tech Support

Original Source

DOI: https://doi.org/10.3390/mi14061134

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