NB slides are coated uniformly with trillions of NanoCone, a cone-shaped organic compound. The apex of NanoCone is functionalized with a NHS (N-hydroxyl succinimidyl) or aldehyde group for directly attaching a protein molecule. Coating with NanoCone provides controlled regular spacing between the proteins to minimize the steric hindrance and to enhance the binding kinetics. Therefore, microarrays fabricated on the NB slides provide both high specificity and sensitivity, and small amount (30~50 μg) of total protein extract is required. Out of two different NanoCone slides currently provided, NB9 slide is suitable for the oligonucleotide microarray due to lateral spacing of 3 to 4 nm, while NB27 slide with the lateral spacing of 6 to 7 nm is ideal for the protein microarray. To confirm outstanding features of the protein microarray, the performance is compared with that fabricated on a commercially available hydrogel slide. It is remarkable to note that the former microarray results in higher signal intensity in a wide range of antigen and antibody concentrations (See the figure in the below). The efficacy saves researcher‘s experimental cost by decreasing the amount of valuable antibody and antigen. Besides, it has been demonstrated that the NB antibody microarray can be stored at -20℃ up to six months.
Figure 1. Protein microarray sensitivity. In this comparison study a total serum protein was labeled with Cy3. The labeled protein was used for the hybridization on the antibody microarray. After the antibody was diluted with a printing buffer to make 0.35, 0.20, 0.10, and 0.05 mg/ml, and a contact microarrayer was employed for the spotting. After adding the sample, an Agilent hybridization chamber was placed in a hybridization oven for 2 hours at room temperature. The fluorescence image was acquired with a confocal laser scanner (GenePix personal 4100A from Molecular Devices), and the signal was analyzed with GenePix Pro 6.0 software (Molecular Devices). For fair comparison, the protocol recommended in company’s user guide was followed.
384 spots of 96 different antibodies, use of a serum protein-Cy5 (8.0 μg)
Figure 2. Fluorescence images after the hybridization with a total serum protein of 8.0 μg.