Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer

Abstract

The interest in fast and sensitive assays for proteases, that is, enzymes that specifically cleave peptide bonds, has increased considerably in the last few years. Two medically important facts in particular have accelerated the development of proteolytic assays. One is that proteases are implicated in more and more diseases. Because of their involvement in tumor progression and metastasis, for example, matrix metalloproteinases, urokinase plasminogen activator (uPA), and cathepsins such as cathepsin B and cathepsin D, proteases play a central role in cancer diagnosis and follow-up of malignant diseases. [1][2][3][4][5][6] In addition, viral infections such as HIV could be detected directly by detection and monitoring of their own specific proteases. This underscores the need for new highly sensitive and fast assays for the specific detection of proteolytic enzymes. [7][8][9][10][11] To date, several different fluorescence-based assays have been developed to prove the presence of a specific protease in a sample using labeled enzyme substrates. Typically, enzyme substrates, for example, a specific peptide sequence, are doubly labeled with a donor and an acceptor fluorophore in a way that ensures efficient fluorescence resonance energy transfer (FRET). [12,13] Upon cleavage of the peptide substrate by a protease the interaction between the donor and acceptor fluorophore is lost. This enables the direct monitoring of protease activity by measuring the increase in donor fluorescence intensity. Alternatively, a peptide substrate might be doubly labeled with the same fluorophore. In aqueous solution hydrophobic interactions of the fluorophores might force the peptide into a conformation in which the fluorophores can form non-or only weakly fluorescent dimers. [14][15][16][17] Nevertheless, chemical modifications of peptide substrates reduce the affinity of the resulting probe to the target molecule and hence the detection sensitivity of the assay. Thus, a minimum of chemical modification is favorable for the design of high-affinity molecular probes for proteases. In addition, doubly labeled peptide probes require site-specific labeling with two extrinsic fluorophores, and synthesis of such compounds is still relatively complicated and expensive. Furthermore, incomplete labeling of peptide probes with only one extrinsic fluorophore, which is not quenched, complicates highly sensitive assays because of the stronger background signal. A method that takes advantage of properties of naturally occurring amino acids instead of a second extrinsic fluorophore would be more useful.Here we describe a novel fluorescence assay for the ultrasensitive and specific detection of proteolytic enzymes in homogeneous solution using singly labeled peptide probes, the fluorescence of which is quenched by the substrate itself. The basic idea of the experiment is that the fluorescence of suitable fluorophores is efficiently quenched by the amino acid tryptophan by means of photoinduced electron transfer (Figure 1). [18][19][20][21][22][23] All...

Photoinduced Electron Transfer (PET) Reactions

“…In the past PET sensors or probes have been developed that use conformationally induced alterations in PET efficiency upon binding, for the specific detection of DNA or RNA sequences, antibodies, and also proteases and nucleases at the single-molecule level [29,32,33,37,38,83,117]. In the past PET sensors or probes have been developed that use conformationally induced alterations in PET efficiency upon binding, for the specific detection of DNA or RNA sequences, antibodies, and also proteases and nucleases at the single-molecule level [29,32,33,37,38,83,117].…”

“…Here again, contact-induced fluo-rescence quenching via PET between fluorophores and the amino acid tryptophan or DNA base guanine emerge as methods for ultra-sensitive and specific detection of protease and nuclease enzymes in homogeneous solution (Figure 7.10) [117]. Here again, contact-induced fluo-rescence quenching via PET between fluorophores and the amino acid tryptophan or DNA base guanine emerge as methods for ultra-sensitive and specific detection of protease and nuclease enzymes in homogeneous solution (Figure 7.10) [117].…”

“…Here again, contact-induced fluo-rescence quenching via PET between fluorophores and the amino acid tryptophan or DNA base guanine emerge as methods for ultra-sensitive and specific detection of protease and nuclease enzymes in homogeneous solution (Figure 7.10) [117]. Thirdly, the assay enables fast, specific, and highly sensitive detection of enzymes with a broad dynamic range (more than six orders of magnitude), and detection limits below the picomolar range (Figure 7.10) [117]. Firstly, the synthesis of large quantities of quenched probes is uncomplicated and inexpensive compared with the use of doubly labeled probes.…”

Photoinduced electron transfer (PET)-probes for the study of enzyme activity at the ensemble and single molecule level

“…With careful design efficient single-molecule sensitive PET sensors can be produced. If quenching interactions between the fluorophore and dG or Trp residue are deteriorated upon specific binding to the target, for example due to binding of a complementary DNA sequence or antibody or due to cleavage by an endonuclease or protease enzyme, fluorescence of the sensor is restored [3,[5][6][7][8][9][10][11].…”

A New Polymeric pH Sensor Based on Photophysical Property of Gold Nanoparticle and pH Sensitivity of Poly(sulfadimethoxine methacrylate)

“…[1][2][3][4][5][6][7][8][9][10][11][12] In particular, there have been many attempts to use optical signals of the nanosized functional materials as a transducer for detecting many important biological and environmental changes, since the change of optical signal has been regarded as a convenient and facile way to monitor those phenomena. For this reason, quantum dot and gold nanoparticle (AuNP) have been widely used as one of the promising candidates for the purpose.…”