Comparison of Oligonucleotide Immobilization Methods for Paper‐Based Diagnostics, With a Focus on Nucleic Acid Lateral Flow Assays

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Introduction One of the main success factors in methods such as lateral flow assay (LFA) is the binding of special reagents, such as absorption probes or antibodies to the nitrocellulose membrane surface, which depends on the immobility and stability of these reagents on the membrane. The aim of this study is to compare different immobilization methods of nucleotide probes, including baking of nucleotide probes, UV cross‐linking, and streptavidin/biotin immobilization on nitrocellulose membrane. Materials and Methods After preparation of nitrocellulose membrane, the dotted probes washed with 4x SSC buffer after exposure to UV (125 mJ/cm 2 ), heat, and the streptavidin–biotin method. Next, the solution was collected and read with a nanodrop. Also, the rolling circle amplification (RCA) method was used to check the fluorescent intensity (aminoallyl‐UTP‐X‐Cy5) in the remaining probes on the surface of nitrocellulose at the wavelengths of 643–670 nm. Results The results of this study show that 60 s‐UV and 2 h‐heat showed the lowest amount of probe removal and wastage after washing in reading with nanodrop and RCA and aminoalyl‐UTP‐X‐Cy5 method. Conclusion Our study showed the best technique to achieve the optimal binding of the probe to the nitrocellulose membrane in LFA and other methods of needing to spot the probe on this type of membrane.

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