Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K(ass.), ranging from 96 to 206 M(-1). Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.

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...

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