Table 3. Typical Procedures of Coupling Strategies with SAM-Modified Au Surfaces.

entry	linker	coupling strategy	Cca ($)	typical procedureb	benefits	risks
a	HS–(CH2)n–COOH, i.e., MHDA	EDC/NHS, coupling with lysine residues	165	gold slides are incubated overnight 250 μM of MHDA in EtOH and activated by incubation in 2 mM EDC/5 mM NHS solution in MES buffer; after rinsing in PBS, 50 μg/mL of IgG in PBS is added for incubation (30 min, room temperature)	• low cost	• water and air sensitive
						• multiple steps required
						• random orientation
b	HS–(CH2)n–CHO	coupling with lysine residues	27	the −NH2 gold chip is immersed in a 5% glutaraldehyde solution in PBS (2 h at room temperature); after PBS rinsing and drying, the chip is incubated with a 100 μg/mL solution of IgG in PBS for 1 h at room temperature	• low cost	• multiple steps required
					• good stability	• random orientation
c	DTSSP	coupling with cysteine or lysine residues	3000	gold chip is incubated in a 1.5 mM solution of DTSSP in PBS for 1 h at room temperature; after rinsing, slides are incubated with 40 μg/mL of IgG in PBS solution for 30 min at room temperature	• short time	• water and air sensitive
						• high cost
						• random orientation
d	CS2/NH2–(CH2)n–NH2	coupling with lysine residues	0.08	gold slides are immersed in a mixed solution (1:1, v/v) of 0.1 M CS2 in water and 20 μg/mL of IgG in PBS solution for 2 h at 4 °C	• low cost	• random orientation
					• short time
					• good stability
e	HS–(CH2)n–NH2, i.e., cysteamine	coupling with acid residues activated by EDC/NHS	40	1 mg/mL solution of IgG is activated by incubation in 2 mM EDC/5 mM NHS solution in MES buffer (15 min at r.t.); after quenching using 2-mercaptoethanol (1.2 μL) and purification by desalting column, the solution in PBS is used for incubation (30 min, room temperature) of the functionalized gold chip	• low cost	• water and air sensitive
					• right orientation	• multiple steps required
						• Ab clustering

^aCalculated as the sum of commercial costs per gram or per milliliter of all the required reagents, from the Sigma–Aldrich product catalog.

^bInspired by refs (48, 62, and 69−72).