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 .
Emgc10
Manufactured by BBI Solutions
Sourced in United Kingdom
The EMGC10 is a compact and versatile electrophoresis power supply designed for a wide range of electrophoresis applications. It provides a stable and consistent power output to ensure reliable and reproducible results. The device features a clear digital display, intuitive controls, and safety features to ensure a safe and efficient operation.
Automatically generated - may contain errors
2 protocols using emgc10
1
SH-SAW Biosensor for CRP Detection
2
Cryo-Ultramicrotomy for Vitreous Sections
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Vitreous sections were cut using the strategy proposed by Ladinsky (2010 (link)). Frozen-hydrated samples were cut using a Leica UC7/FC7 cryo-ultramicrotome (Leica Microsystems, Vienna, Austria) at −150°C. First a 100 × 100 × 30–μm mesa-shaped block was trimmed using a Trimtool 20 diamond blade (Diatome, Hatfield, PA). Sections were then cut using a Cryo 25° diamond knife (Diatome). The nominal thickness was set to 50–100 nm and cutting speed to 1 mm/s. A customized micromanipulator (MN-151S, model EDMS12-260; Narishige, Tokyo, Japan) was used to control the cryoribbon. A Crion electrostatic charging device was operated in discharge mode to prevent the sections from sticking to the diamond blade (Pierson et al., 2010 (link)). Once the ribbon was 2–3 mm long, it was transferred onto an EM grid (Protochips C-flat [Protochips, Morrisville, NC] or EMS continuous carbon grid, CF-200-CU-50 [EMS, Hatfield, PA]) to which 10-nm gold fiducials (EM.GC10; BBI Solutions, Cardiff, UK) were already added. This transfer was initiated by operating the Crion in charge mode. Subsequently, the ribbon was physically pressed with a 10-mm laser window glass (65-855; Edmund Optics, Barrington, NJ) to ensure firm attachment. The grid was stored in liquid nitrogen until imaging.
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