Carboxylic acid-SAM Formation ReagentC488 Product Manual

Carboxylic acid-SAM Formation Reagent is applied for the preparation of carboxylic acid terminated self-assembled monolayers (SAMs) on a gold surface of biosensors such as for QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance) or electrical analysis. Carboxylic acid-SAM can be used as an interface to immobilize a protein or a peptide on a gold surface coupled with carboxyl group activation method using NHS/WSC. Amine Coupling Kit (Product code: A515) is available for protein immobilization on the gold surface coated with a carboxylic acid-SAM.
Carboxylic acid-SAM Formation Reagent is especially designed to minimize a non-specific protein adsorption on the surface.

Fig.1 Preparation of a biosensor using Carboxylic acid-SAM Formation Reagent

Carboxylic acid-SAM Formation Reagent

1 μmol x 3

Since a trace amount of colorless or slightly yellowish liquid is in the tube, please centrifuge prior to use.

0-5 ℃

Preparation of a Carboxylic acid-SAM surface on a gold substrate

1. Add 1 ml ethanol to a tube, and pipette to prepare 1 mmol/l Carboxylic acid-SAM solution. ^*1 Then, dilute the solution 10-fold with ethanol for Step 2).
2. Immerse a gold substrate ^*2 in the reagent solution prepared at Step 1) at room temperature and leave it overnight.
3. Wash the substrate several times with ethanol and purified water sequentially. ^*3

1. If the reagent hardly dissolve by pipetting, use an ultrasonic bath. Use freshly prepared reagent solution at Step 2).
2. Clean the gold substrate with Piranha solution, if necessary. Mix 3 volumes of sulfuric acid and one volume of hydrogen peroxide solution to prepare 　Piranha solution.Since the solution is severely caustic, handle with great care.
3. Store the SAM-coated substrate under nitrogen gas in a tightly sealable glass container at 0-5 ℃.

This product does not include necessary reagents and solutions for a protein immobilization. Amine Coupling Kit (Product code: A515) is available for the protein immobilization on the carboxylic acid-SAM surface. If not use the kit, follow this general protocol.

Required reagents and buffers

• N-Hydroxysuccinimide (NHS)
• 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC)
• pH4-6 buffer solution
  • MES buffer is generally used.
• pH8-9 buffer solution
  • Carbonate buffers, phosphate buffers or good's buffers are generally used.

1. Prepare 100 mmol/l NHS and 100 mmol/l WSC solutions using pH4-6 buffer solution .
2. Mix equal volume of NHS and WSC solutions immediately, and add to the carboxylic acid-SAM coated gold substrate.
3. Leave it at room temperature for 1 hr, and wash several times with pH4-6 buffer solution.
4. Add a protein solution ^*1 to the substrate, and leave it at room temperature for 1 hr.
5. Wash with PBS, and go to blocking process with a blocking solution ^*2 if necessary.

1. The solution should be prepared with a buffer solution (pH8-9). Use 10-1,000 μg/ml of protein solution for immobilization.
2. One mol/l ethanolamine solution (pH8.5) is commonly used for blocking.