Emgc30

Colloidal suspension of 40 nm AuNPs (EMGC40; BBI Solutions) or 30 nm AuNPs (EMGC30; BBI Solutions) was modified with streptavidin, as described previously with small modification (24 (link)). They were suspended in 10 mM phosphate buffer (pH 8.0) with 0.2% Tween 20. To form biotin-functionalized surface on AuNP, alkanethiol mixture solution was added at a final concentration of 0.1 mg/mL biotinylated alkanePEG thiol (SPT-0012D; SensoPath Technologies, Bozeman, MT), 0.2% carboxy-EG6-undecanethiol (C445; Dojindo), and 0.2% hydroxy-EG6-undecanethiol (H355; Dojindo). After incubation for 6 h at 70°C, unreacted alkanethiols were removed by centrifugation (10,000 × g, 3 min for 40 nm AuNP and 5 min for 30 nm AuNP) and mixed with streptavidin (PRO-791-b; Prospec, Fullerton, CA) at a final concentration of 0.67 mg/mL, dissolved in 10 mM phosphate buffer (pH 8.0) with 0.2% Tween 20. The solution was gently mixed using a rotator for 3 h at room temperature, and unreacted streptavidin was removed by centrifugation (10,000 × g, 3 min for 40 nm AuNP and 5 min for 30 nm AuNP). The pellet was suspended in 10 mM phosphate buffer (pH 8.0) and stored in a refrigerator at 4°C. Just before starting measurement, biotinylated kinesin-1 and streptavidin-coated AuNP were mixed at a ratio of 2 (kinesin-1 molecules/AuNP particles).

A sandwich assay to detect C-reactive protein (CRP) was employed to evaluate SH-SAW sensor measurement characteristics in specimens with different molecular sizes. The sandwich assay was performed by reacting with targeting proteins. In the sandwich assay, we used CRP with capture antibodies. Subsequently, secondary antibodies were used for amplifying the sensor output signals reacted with the CRPs captured in the previous step. The capture antibodies were immobilized onto the sensing area of the SH-SAW biosensor using a crosslinking chemical (dithiobis[succinimidylpropionate]), DSP, #22585, Thermo Scientific, Waltham, MA, USA). The HyTest (Turku, Finland) 4C28-CRP30 monoclonal antibody was used as the capture antibody. We used recombinant CRP (CRP Calibrator L-710, SHINO-TEST CORP., Tokyo, Japan) in a series of measurements. The HyTest 4C28-CRP135 monoclonal antibody was used as the secondary antibody. The secondary antibodies were conjugated with gold nanoparticles with diameters of D = 10, 15, 20, and 30 nm (EMGC10, EMGC15, EMGC20, and EMGC30, BBI Solutions, Crumlin, UK) to vary the molecular size. A structural diagram of the antigen, antibody, and gold nanoparticles on the SH-SAW biosensor surface is shown in Figure 3.