TAMRA fluorophore

Since some DNAzymes depend on specific metal ions for activity, they can be employed for the detection of those ions, hi the first example, a previously reported [59] DNAzyme was labeled with a Dabcyl fluorescence quencher at the 3 -end and the corresponding RNA substrate with a TAMRA fluorophore at the 5 -end [60]. Upon addition of Pb " ions, the substrate was cleaved, resulting in dissociation from the DNA enzyme strand. This led to spatial separation of the fluorophore-quencher pair, resulting in fluorescence (Fig. 5). The sensor system was over 80 times more responsive to Pb than to other metal ions, and had a quantifiable detection range of 10 nM to 4 pM. A similar strategy was developed for the detection of Ctf by a DNAzyme that oxidatively cleaves DNA [61]. The system showed a dynamic range of 35 nM to 20 pM and had a metal ion selectivity of a factor of 2000 for Cu over other metal ions. A comparable system was reported for the detection of the uranyl cation (UO/ ), with millionfold selectivity over other metal ions and parts-per-trillion sensitivity. 

This approach has been shown to work with a number of different fluorescent probes such as the short-wavelength fluorophores dansyl sul-fonyl chloride and coumarin chloride and the long-wavelength fluorophores tetramethylrhodamine-5-(and-6)-isothiocyanate [5(6)-TRITC], 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester [5(6)-TAMRA, succin-imidyl ester] and lissamine rhodamine B sulfonyl chloride (each in conjunction with different binding functionalities on the SAM surface. 

An amino-functionalized TPEDA SAM was synthesized onto a glass surface as described previously. Then a PDMS stamp previously inked in an acetonitrile solution of the fluorophore (TAMRA or lissamine) was brought into contact with the SAM for a few seconds, resulting in the covalent attachment of the fluorophore to the layer. The slide was subsequently immersed in an acetonitrile solution of a reactive molecule for the attachment of the binding groups onto the surface (i.e., urea or amide) at the sites of the unreacted surface amino groups (Fig. 10). 

All data obtained with Tecan Ultra Evolution MTP reader. The following excitation and emission wavelengths were used EDANS and AMC 350 and 500 nm RhllO 485 and 535 nm TAMRA 535 and 595 nm PT14 405 and 450 nm. 4 = primary cleavage site confirmed by MS. AMC = aminomethylcoumarin. RhllO = rhodamine 110. yE = glutamic acid attached to RhllO via its carbonic acid in side chain. EDANS = fluorophore 5-[(2-aminoethyl)amino]naphthalene-l-sulphonic acid. DABCYL = 4-(4-dimethylaminophenylazo)benzoic acid quencher. BTN = biotin. PT14 = acridone-based fluorescence lifetime label. 

Practically, the g factor can be determined using a free fluorophore with a known anisotropy value as a standard. Free fluorophores like TAMRA (5,6-carboxytetramethylrhodamine) and fluorescein with rtme values of rtrue = 0.0202 (ptme = 30 mPU, see below) and rtrue = 0.0134 (ptrue = 20 mPU, see below), respectively, can be used (some sources report 27 mPU for fluorescein in solution) ... 

Fig. 4.13 Top Schematic representation of the monolayer library synthesis (TM0-TM6, T0-T6, L0-L6) in a microtiter plate. Three different acetonitrile solutions containing each a different fluorophore, TAMRA (TM), TRITC (T), or Lissamine (L) are pipetted in the wells of three consecutive columns of a MTP coated with TPEDA monolayer (A in this figure). Subsequently, six solutions (1-6) containing each a different ligand are pipetted in consecutive rows. No ligand solution is added on the top row (wells TMO, L0, TO). Bottom. Chemical composition of the library of fluorescent SAMs (TM0-TM6, L0-L6, T0-T6) prepared on the MTP glass surface 58. Reproduced with permission...

Figure 7 Peptide sequences and chemical structures of amino acids and fluorophores. Shown are the sequences for peptides TBQ, TH2, TFl, and TF2 and structures of y3-alanine (single amino acid code B), the fluorescence donor NBD, and acceptor TAMRA. Leucine residues replaced by hexafluoroleucine in the peptide sequences are indicated in green.

The present electrocyclization protocol is also applicable to rapid fiuorescent labeling 10-100 pg of albumin and anti-GFP mAb were labeled with coumarin lb and TAMRA probes Ic, respectively (entries 8 and 9). As little as 2 pg of anti-GFP antibody ( 10 pmol) was successfully labeled by Ic in 30 min (entry 10) 20 equiv. of Ic preferentially labeled the more accessible Fc fragment with two fluorophore molecules, while retaining its GFP recognition activity with 90% of the intact mAb. 

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