Fluorescent Macromolecular Sensors of Enzymatic Activity for In Vivo Imaging - Progress in Molecular Biology and Translational Science - page 366

A C

80

0.45

70

0.40

60

0.35

+trypsin

0.30 50

- T +T

0.25

40

0.20

30

0.15

20

0.10

10

0.05 - trypsin

0.00 0

640 660 680 700 720 740

500 600 700 800 900 1000

Wavelength (nm)

Wavelength (nm)

C

0.25

D

2500

0.20

2000

0.15

1500

0.10

Tr y p s i n

1000 a CT

0.05

500

0

0.00

2 468 0

Time (min)

12 14 16 18

500 600 700 800 900 1000

Wavelength (nm)

Figure 9.4 Spectral properties of PGC-based enzyme-sensitive probes. (A) Absorbance
spectra of IRDye 800CW dye linked to PGC at high conjugation density (gray trace) and
low conjugation density (black trace). (B) Same as A but after 30 min incubation in the
presence of trypsin. Arrows are pointing at the blue-shifted peak of H-dimers of IRDye
800CW dye. (C) The change of excitation (ex) and emission (em) spectra of PGC-Cy5.5
before and after the addition of trypsin. The corresponding effect on detectable red
fluorescence of Cy.5.5 is shown in the inset. (D) Experimental demonstration of PLL-
backbone encoded specificity: the addition of CT) does not result

in PGC-Cy5.5 cleavage, whereas trypsin results in rapid degradation of PGC-Cy5.5 mol-
ecules and fluorescence intensity increase.

a -chymotrypsin ( a

degraded by nonspecific esterases and also quickly fall apart in alkaline or
acidic media. They usually result in efficient and complete degradation of
PGC probes into small fragments and result in the highest relative changes
of fluorescence intensity and lifetime. Overall, FL changes are greater for
cyanine dyes that fluoresce in the far-red range of the spectrum; that is,
Cy5.5, IRDye 680RD produce a very profound change of FL (up to fivefold)