Methylene blue modification is a post synthesis conjugation to a primary amino group. The amino group can be placed at the 5' and 3' and for internal positions an amino modified base is used, e.g Amino dT C6
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.
~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis.
Methylene Blue (e.g Atto MB2) NHS is a redox-active, heterocyclic aromatic dye that, when incorporated at the 5' or 3'-end of an oligonucleotide, enables the modified oligo's use as an electrochemical (EC) probe for nucleic acid analysis. Currently, there is considerable interest in using MB-modified oligonucleotides as aptamer probes for developing electrochemical DNA sensors for selective and sensitive detection of specific biochemical targets (DNA, RNA, proteins, etc.) in complex samples (for example, blood serum) (1,2). Such sensors are constructed by covalent attachment (typically through one or more thiol groups) of the MB-modified DNA probes to the surface of a gold electrode. The binding of target to probe leads to changes in the structural dynamics of the probe DNA that change the distance between the MB moiety and the gold surface. For "signal-on" sensors, the MB moiety moves close enough to the gold surface to cause electron transfer between the two, and generation of an electrochemical signal indicating presence of target in the sample (3). For "signal-off" sensors, the MB moiety moves away from the gold surface, halting electron transfer between the two, with the subsequent loss of an electrochemical signal indicating presence of target in the sample (4). Intensive work continues to move these systems beyond proof of principle and towards commercial availability.
1. Ricci, F., Lai, R.Y., Plaxco, K.W. Linear, redox modified DNA probes as electrochemical DNA sensors. Chem. Comm. (2007), 36: 3768-3770.
2. Song, S., Wang, L., Li, J., Zhao, J., Fan, C. Aptamer-based biosensors. Trends in Anal. Chem. (2008), 27: 108-117.
3. Ferapontova, E.E., Gothelf, K.V. Optimization of the Electrochemical RNA-Aptamer Based Biosensor for Theophylline by Using a Methylene Blue Redox Label. Electroanalysis (2009), 21: 1261-1266.
4. Xiao, Y., Lubin, A.A., Heeger, A.J., Plaxco, K.W.. Label-free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-Based Sensor.Angew. Chem. Int. Ed. Engl. (2005), 44: 5456-5459..