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t1   Fluorescence Lifetime Applications t1
 
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Fluorescence Lifetime (FLT) Probes and Labels
  1. Environment-sensitive lifetime dyes
        
 

Selected Square and Seta dyes exhibit shorter fluorescence lifetimes (FLTs) in aqueous media or when labeled to low molecular weight analytes (drugs or small ligands) but increased brightness and up to ten-times longer FLTs when bound to proteins or oligonucleotides. These environment sensitive Seta and Square labels are well-suited as tracers in FLT-based assays, where e.g. the FLT-tracer would be labeled to a small antigen (ligand) displaying a short lifetime but showing a substantial FLT-increase upon binding to a larger protein (antibody or receptor).  K8-1300 is also used as a fluorescent probe that binds to hydrophobic regions in cells.  

The table below provides data of the expected changes in lifetime for selected FLT dyes. It is important to note that the observed lifetime change of a fluorescently labeled ligand will strongly depend on the nature and size of the biomolecule it binds to:
Product Dye Dye–Conjugate Δτ[ns]
 τ(Dye–conj.) – τ (Dye)
τmean [ns]D/P ratioτmean [ns]
0.46
0.96
3.2
2.80
0.29
1.2
3.3
3.09
0.7
1.0
3.0
2.70
0.25
1.0
2.8
2.56

 
 

SETA BioMedicals offers several of these environment-sensitive FLT labels: all are excitable with a 633, 635 or 647 nm laser source.

a) FLT probes

Probes such as Square-670 (K8-1300) show large changes in FLT upon binding to either proteins or cell-surfaces. K8-1300 is water-soluble but with a strong affinity for the hydrophobic regions of biomolecules.
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Comparison of the frequency responses of Square-670 (K8-1300)
before and after binding to protein
(τfree = 460 ps; τbound = 3.26 ns)		 Comparison of the intensity decays of Square-670 (K8-1300)
before and after binding to protein
(τfree = 460 ps; τbound = 3.26 ns)
 

ProductPack SizeProduct Code
1 mg
K8-1300-1mg

Pricing and Availability
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    Square 650-pH is a NIR probe that exhibits a pKa around 7 and is therefore useful for measurement of pH changes in the physiologically relevant pH range from pH 6 to pH 8. Importantly both forms (protonated as well as unprotonated) of Square 650-pH are fluorescent and they exhibit different lifetimes which makes this compound useful for lifetime based applications (see Figure below). For more information about these dyes we refer you to their specs sheets and the published literature.

    pH-ProbepHτ1f1

    Square-650-pH    		 2.0 1.17 1.0
    9.0 0.53 1.0
    ProductPack SizeProduct Code
    1 mg K8-1407-1mg
    1 mg K8-1405-1mg
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    Frequency responses of Square-650-pH (K8-1407)
    at acidic and basic pH.

    		 Phase angle changes vs. pH for Square-650-pH
    measured at 100 MHz.
     
    b) Hydrophilic FLT labels
    These are reactive labels that show changes in FLT upon binding to either proteins, oligonucleotides or cell-surfaces. In general they are labeled to a low-molecular-weight binding partner and interaction with a high-molecular-weight protein or receptor leads to a change in FLT:
     
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    Comparison of the frequency responses of Seta-633 (K8-1663)
    before and after binding to protein or oligos.
    (τfree = 250 ps; τbound = 2.8 ns)

    		Comparison of the intensity decays of Seta-633 (K8-1663)
    before and after binding to protein or oligos.
    (τfree = 250 ps; τbound = 2.8 ns)
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    Comparison of the frequency responses of Seta-660 (K8-1352)
    before and after binding to protein or oligos.
    (τfree = 290 ps; τbound = 3.3ns)

    		 Comparison of the intensity decays of Seta-660 (K8-1352)
    before and after binding to protein or oligos.
    (τfree = 290 ps; τbound = 3.3 ns)
     

 

    Applications:

    Below is a list of fluorescence lifetime (FLT) based reagents suitable for the measurement of small molecular weight antigens as described in: Bioconjugate Chem. 20, 1807–1812 (2009). Related products are listed in the following table:

     

      Examples of lifetime based assays:


    – Amino-peptidase activity measurements
    – Caspase-activity measurements



    2. Environment-insensitive lifetime labels

     
    a) Long-lifetime, short-wavelength FLT labels

    SETA BioMedicals     also offers long lifetime labels with lifetimes in the range between 32 and 9 ns. These dyes are excitable in the UV or blue region and are useful as polarization probes for the measurement of high MW analytes (small proteins).
    SeTau-425-NHS (K7-545) for example was used to measure high molecular-weight analytes such as BSA directly in a FP assay. Importantly the FLT of these labels does not change upon labeling or binding to proteins or analytes. These labels exhibit long lifetimes that are not environment sensitive.
     
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    CompoundLifetime [ns]
    SeTau-380-NHS (K1-204) 32
    SeTau-404-NHS (K7-549) 9
    SeTau-405-NHS (K7-547) 9
    SeTau-425-NHS (K7-545) 26
    ProductPack SizeProduct Code
    SeTau-380-NHS 1 mg K1-204-1mg
    SeTau-404-NHS 1 mg K7-549-1mg
    SeTau-405-NHS 1 mg K7-547-1mg
    SeTau-425-NHS 1 mg K7-545-1mg
    		 
    Pricing and Availability
    Ordering
     

    b) Long-wavelength, squaraine-rotaxane-based FLT labels

    These reactive labels are excitable in the red and NIR region and are useful as polarization probes for the measurement of low MW analytes. SeTAU-655-NHS, SeTau-647-NHS or SeTAu-665-NHS are examples of FLT labels with lifetimes that are insensitive to the environment. The average lifetime of these labels is in the order of 2.5 – 4 ns which make them superior lifetime or polarization probes over Cy5 or Alexa dyes (Cy5, Alexa 647: lifetime ~ 1 ns in water). The longer lifetimes make them also more suitable donors in energy-transfer-based assays assuring that even at high energy transfer rates the lifetimes of these labels is not too short to be accurately measured with inexpensive instrumentation.
     
    ProductLifetime [ns]
    SeTau-647-NHS
    		 3.2
    SeTau-665-NHS
    		 3.1
    ProductPack SizeProduct Code
    SeTau-647-NHS 0.5 mg K9-4149-0.5mg
    SeTau-665-NHS 0.5 mg K9-4119-0.5mg
    Pricing and Availability      
    Ordering      
     

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Frequency responses of SeTau-665 (K8-4119) in
aqueous buffer solution pH 7.4.

Intensity decays of SeTau-665 (K8-4119) in aqueous
buffer solution pH 7.4 in comparison to Cy5 and Alexa 647.

 
 
 
Product Specifications:
Excitation Light Sources Free Dye Dye Conjugate
Product Number Product Name 380 405 436 488 532 594 635 670 Solvent λAb max. [nm] ε [M–1
cm–1]		 λEm max. [nm] QY [%] FLT [ns] Carrier
PB 7.4		 λAb max. [nm] λEm max. [nm] QY [%] FLT [ns]
                           
K7-545 SeTau-425-NHS • 425 4200 545 39 26.2 0.0 427 528 16
K7-547 SeTau 405 NHS • • PB 7.4 405 13800 518 80 9.3 BSA, D/P=8 406 518 20 7.6
K7-549 SeTau-404-NHS • • PB 7.4 402 10600 515 52 9.3 BSA, D/P=2 402 503 8.8
K8-1342 Seta-670-NHS • • • PB 7.4 667 179000 688 7 BSA, D/P=1 681 695 35 2.4
K8-1352 Square-660-NHS • • PB 7.4 658 182000 677 3 0.26 BSA, D/P=1 676 695 13 3.32
K8-1500 Square-655 • • • MeOH 628 177000 654 0.09 1 g/L BSA 652 672 80
K8-1626 Seta-633-di-NHS • PB 7.4 635 192000 644 12 0.7 IgG, D/P=1 639 650 33 3
K8-1663 Seta-633-NHS • PB 7.4 633 250000 644 7 0.3 IgG, D/P=1 637 647 26 1.8
K8-1669 Seta-635-NH-NHS • PB 7.4 638 136000 655 31
K8-1672 Seta-646-NHS • PB 7.4 646 207000 656 10 IgG, D/P=1 650 661 33
K8-1682 Seta-660-di-NHS • PB 7.4 661 151000 672 11 IgG, D/P=1 665 675 49
K9-4112 SeTau-655-di-NHS • PB 7.4 665 200000 673 25 0.9 IgG, D/P=1 657 675 31 1.8
K9-4113 SeTau-665-di-NHS • • PB 7.4 665 140000 715 62 3.1 IgG, D/P=1 664 709 43 3.3
K9-4142 SeTau-647-di-NHS • PB 7.4 650 368000 694 65 3.2 IgG, D/P=0.5 649 691 58 3.2
K1-204 SeTau-380-NHS • Water 270 23800 480 56 32.5 BSA, 383 468 44 30.6
K8-1344 Seta-665-NHS • • • PB 7.4 667 118000 683 3 BSA, D/P=1 682 697 38 3
K8-1367 Seta-670-NH-di-NHS • • PB 7.4 672 93000 696 15 2 IgG, D/P=1 684 703 7
K8-1377 Seta-690-NH-di-NHS • • PB 7.4 693 113000 714 14 BSA, D/P=1 711 726 14 1.5
K8-1762 Seta-635-B-di-NHS • • • • PB 7.4 634 131000 656 8 0.5 BSA, D/P=0.5 650 672 41 3
K8-1782 Seta-660-B-di-NHS • • • • PB 7.4 659 132000 679 9 BSA, D/P=1 672 695 28 2.2
K8-1667 Seta-635-NH-di-NHS • PB 7.4 640 188000 656 33 1.8 IgG, D/P=1 646 663 21
K8-1300 Square-670-di-COOH • • • PB 7.4 667 207000 685 7 0.46 6g/L BSA 678 698 28 3.2
K9-4149 Seta-647-NHS • PB 7.4 649 200000 695 61 3.2 IgG, D/P=1 648 694 59 3.3
K9-4119 SeTau-665-NHS • • • PB 7.4 664 160000 712 53 3.1 IgG, D/P=1.2 653 716 50 3.3
K8-1407 Square-650-pH-NHS • PB 7.4 653 135000 671 16 1.17 IgG, D/P=1 662 677 7 1.52
 
 
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