Practical steps for glass utensils cleaning, reagents set-up, RNG synthesis and immobilization as substrate for Raman spectroscopic sensors and biosensors
Prior to the synthesis step the operator must consider the utensils cleaning and the reagents solution preparation. The user is recommended to perform this work in a well-ventilated area such as a fume cupboard. Ensure that the operator has appropriate personal protective equipment to include goggles, appropriate gloves and a lab coat.
Step 1. Vessels cleaning
The glass vessels cleaning and drying is essential for a successful synthesis. The glass utensils involved in gold synthesis are cleaned by keeping them for 30 minutes in aqua regia solution HClconc: HNO3 conc (v:v 3:1). Afterwards the glasses are washed with water, ethanol, acetone, and deionized water and kept in a dry box at 37°C. The glassware is next heated at 160-170°C for one hour.
Step 2. Reagent solutions
For synthesis two solutions are made in a fume-cabinet; a gold salt solution consisting of 0.03 g HAuCl4 to 30 mL distilled water; and a fresh reducing mixture consisting of 0.09 g NaBH4 to 10 mL distilled water. Both solutions are kept on ice for 5 minutes before using them for synthesis (Fig. 1 a-b).
Step 3. RNG synthesis
The gold salt is reduced by hydrogen which is slowly released from NaBH4 in aqueous solution. The synthesis takes place at ice temperature. Pipette 10x100µL of reducing solution to the gold salt solution (Fig. 1 c) under continuous and slow stirring. The magnetic stirring is not recommended. The synthesis is completed when the black color appeared (Fig. 1d). Ensure that the reductive agent is added slowly in volumes of 100 µL to the gold salt. If it is quickly added the long reticules are synthesized; they are not stable in a colloidal state and precipitate in approximate 10 minutes. Close the glassware immediately after the last volume of reductive agent is added, before the excess of hydrogen is out from the system. The dispersion is homogenized by this hydrogen convection and the stirring can be stopped at the last addition of the reductive agent. The colloidal state is assured by NaCl which is formed as additional product during the reaction and act as a valuable surfactant in this case. In the accordance to our one-step synthesis condition one criterion is agreed that no surfactants or other additives need to be added into the dispersion.
Observation: if instead the volume of the reductive agent increased to 2 mL the colloid becomes instable; sediment is formed and the colorless supernatant still contains reticular gold. At lower amount of reductive solution (400 mL, 100 mL, respectively) the shapes become ellipsoidal and spherical. The zeta potential of the colloids was consistently negative (-40 to -50 mV)7.
TIMING: the section steps 2-3 will take to complete maximal 15 minutes.
Pipette suitable aliquot out from stock in a disposable zeta cell and acquire the RNG zeta-potential. Prior to sample splitting for the characterization, the operator checks that dispersion is stable to be able to perform reliable analysis of the RNG. Acquire six replicates to ensure that the sample is stable within a necessary amount of time. If there is evidence of sedimentation in the sample, then the dispersion is not stable enough to allow accurate subsampling.
The colloid can be left up to one month or longer at 4°C.
Step 4. RNG immobilization as substrate for Raman spectroscopic sensor and biosensor
Place the cover slips (18mmx18mm) in a cabinet with oxygen plasma at 0.2 mbar, 110 W for 5 minutes (Fig. 2a). Take out the slide with a pinsetter and place it on a bench. Pipette 100 µL of as prepared colloid on this glass slide (Fig. 2b). Dry it under air pressure (Fig. 2c).
TROUBLESHOOTING the air stream should be perpendicular on the glass surface.