Single molecule detection on a protein-array assay platform                                       

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 three orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, truly quantitative measurements across several orders of magnitude are made possible by this assay configuration, since molecules can be counted individually.
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No particular sample preparation had to be done. Due to excitation via total internal reflection, only molecules near the surface are illuminated. Consequently, unprocessed biological samples (human urine and plasma) could be analysed without the requirement of sample purification or washing steps during analysis. Background generated from light scattering can be separated from the specific signals. The system therefore allows real-time monitoring of the analyte binding event. Samples containing about 600 antigen molecules per microliter produced a distinct signal.
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The technique is widely applicable to the development of new assays in medical diagnostics and research. For analysing the binding of complex, directly fluorescence-labeled protein mixtures to large sets of arrayed antibodies, for example, this technique is highly relevant and important due to its sensitivity, ability to quantify, and analysis of unprocessed samples. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.
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Schmidt et al. (2011) J. Prot. Res. 10, 1316-1322.

A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins                 

Technical advances in single-molecule fluorescence techniques and the ambition of quantification in biological research demand stoichiometrically labelled biomolecules, especially proteins. However, standard protocols are inappropriate for generating stoichiometric labelling, a fact that complicates quantification significantly. To overcome this limitation, a universally applicable labelling and purification process was established to prepare biologically active proteins with a stoichiometric 1:1 ratio of attached dye-label. The dye-label is linked to a specific DNA sequence, which acts as a barcode-like tag for affinity purification. The DNA-dye tag is covalently bound to the target protein, which is present in excess to assure the binding of no more than one dye per molecule. Affinity purification occurs at magnetic beads that are functionalized with oligonucleotides that are complementary to the DNA-tag of the labelled proteins but for one or two mismatches. Washing removes all unbound, unlabelled molecules. The labelled protein is subsequently released by the addition of a fully complementary oligonucleotide. This process allows a gentle purification of a protein fraction that has exactly one label attached to each molecule under conditions that preserve protein structure.

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Friedrich et al. (2011) Proteomics 11, 3757-3760.