Colorectal carcinoma is one of the most frequent and deadliest tumors in the western world. The visualization of cancer-specific enzymatic activities could possibly improve sensitivity and specificity as compared to classical white-light endoscopy. DNase X, which is typically found in early lesions, and TKTL1, which identifies aggressive carcinomas with a high metastatic potential, could potentially constitute such cancer-specific enzymes. Here, fluorescent dyes have been developed in order to specifically detect these enzymatic activities. A fiber-based system was developed for the detection of small concentrations of fluorescent dyes in scattering and absorbing media. With the use of the reflectance spectrum and a theoretical model for the light distribution, the intrinsic fluorescence is assessed from the raw fluorescence. The resulting intrinsic spectrum shows only a weak dependence on the optical properties of the sample and its intensity correlates with the fluorophore concentration. Thus, small concentrations and small variations in the concentrations of the fluorescent dye can be measured. In conclusion, the presented fluorescence diagnostic system in combination with new fluorescent probes has the potential to distinguish between cancerous tissue samples with high enzymatic activity and non-cancerous tissue samples with lower enzymatic activity.
Enzymes engage key roles in a wide variety of important physical and medical processes, which thus can be altered by
manipulating the behavior of enzymes in charge. The capability for manipulation requires an exact understanding of
enzymatic operation modes though, which can be increased by employing fluorescence spectroscopy techniques. To date
several fluorescence-based assays using labeled substrates have been developed to examine different subclasses of
hydrolases. We developed a method that circumvents the unspecific probe enzyme interactions and affinity problems
occurring in common probes as those based on fluorescence resonance energy transfer (FRET) by taking advantage of
the comparably strong electron donating properties of the naturally occurring nucleic acid guanosine (G). Combined with
an appropriate fluorophore this compound shows efficient photoinduced electron transfer (PET) quenching reactions
only upon contact formation. Thus, initially quenched enzyme substrates, e.g. specific nucleic acid sequences, can be
designed that cause a distinct increase in fluorescence signal upon specific hydrolysis. Here we demonstrate the general
validity of PET probes for the observation of various nucleases at the ensemble and single molecule level. The rapid
response time of the probes enables real-time monitoring of enzyme activities and provides quantitative data which are
compared to those of commonly available and recently published, more complex probes. Additionally the applicability
of this method is demonstrated for peptidases via fluorophore tryptophan (Trp) interaction.
Fluorescence based enzyme analysis is commonly done by FRET-probes, natural enzyme substrates flanked by two
corresponding fluorophores, showing spectral changes upon distance variations between the fluorophores. However, the
use of double labeled substrates displays several limitations such as reduction of sensitivity and high background signal
accompanied by high costs for synthesis. Therefore, the development of new probes avoiding these factors is of general
interest in enzyme research. A promising approach represents smart probes, i.e. singly labeled quenched enzyme
substrates that increase fluorescence intensity upon enzymatic cleavage. Smart probes use the fact that certain rhodamine
and oxazine dyes are selectively quenched upon contact formation with guanine or tryptophan residues via photoinduced
electron transfer (PET). The rapid response time of the probes enables real-time monitoring of enzyme activity in
ensemble as well as in single molecule measurements, which is an important prerequisite for the improved understanding
of enzyme mechanisms. We present the design of smart probes for the detection of the two hydrolases, DNaseI and
Carboxypeptidase A (CPA) with respect to stability and substrate specificity in ensemble measurements. Furthermore,
we investigate the influence of the attached fluorophore on hydrolysis efficiency in case of CPA and demonstrate first
applications of smart probes in single enzyme experiments.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.