Counting of Single Protein Molecules at Interfaces and Application of This Technique in Early-Stage Diagnosis

Löscher, Frank; Böhme, Sabine; Martin, John; Seeger, Stefan

doi:10.1021/ac971378r

Cited by 33 publications

(22 citation statements)

References 37 publications

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“…Confocal fluorescence spectroscopy (CFS) has been further developed for the ultrasensitive detection of fluorescent molecules on glass surfaces. Loescher et al [21] described the capture of fluorescently labeled antibodies by a glass surface bound antibody which upon excitation by the laser beam in the confocal detection volume produced single photon bursts over a range of 10 -10 to 10 -17 M that could be clearly separated from a control sample.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Confocal Fluorescence Spectroscopy for the Detection of Pathological Prion Proteins

Dietrich¹,

Bossart²,

Oesch³

et al. 2005

Chimia

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Prion diseases or transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation in the brain of PrP Sc , an abnormal isoform of the host-encoded glycoprotein PrP C . PrP Sc is a reliable marker for the post mortem diagnosis of TSEs but its use as a marker for a pre-clinical blood test has been hampered by the low levels of PrP Sc in blood. We have evaluated confocal fluorescence spectroscopy (CFS) as an ultrasensitive method for the immunological detection of PrP in brain homogenates and blood serum. Using recombinant PrP, we determined the detection limit of our CFS assay to be in the picomolar range which is in the same order of magnitude as the corresponding luminescence immunoassay (Prionics ® -Check LIA). The analytical sensitivity was further investigated using brain homogenates from BSE infected cattle that showed low levels of PrP Sc by Western blot analysis. In these brain homogenates, PrP Sc was detected by confocal fluorescence spectroscopy down to a dilution of 64-fold whereas the luminescence immunoassay was able to detect PrP Sc in brain homogenates diluted down to at least 128-fold suggesting that in this antibody sandwich assay the luminescence immunoassay has a higher analytical sensitivity than confocal fluorescence spectroscopy. We also demonstrated that blood serum is a suitable matrix for a confocal fluorescence spectroscopy based TSE test, and found that serum did not interfere with the detection of spiked recombinant PrP.

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Section: Introductionmentioning

confidence: 99%

Evaluation of Confocal Fluorescence Spectroscopy for the Detection of Pathological Prion Proteins

Dietrich¹,

Bossart²,

Oesch³

et al. 2005

Chimia

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“…By far the most common method for characterizing single molecules in solution as well as immobilized at surfaces is spectroscopy of fluorescently labelled targets. [1][2][3][4][5][6] Furthermore, surface-enhanced Raman scattering has been described as being sensitive to single molecules after they have been attached to a metal nanoparticle. [7] On the other hand, label-free methods like scanning-probe microscopy, including, scanning tunnelling microscopy (STM) or atomic force microscopy (AFM), allow single molecules to be imaged and manipulated on surfaces by using microscopic cantilevers.…”

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confidence: 99%

“…However, single-molecule counting at surfaces can easily be used to identify specific molecular recognition events even at very low concentrations. [2] For this reason, increasing numbers of investigations are being carried out on immobilized single molecules attached to glass surfaces and subsequent scanning with confocal laser-induced spectroscopy. [1,2,5,[11][12][13][14] For binding of capture molecules, different techniques for immobilization onto solid surfaces have been developed.…”

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confidence: 99%

“…[2] For this reason, increasing numbers of investigations are being carried out on immobilized single molecules attached to glass surfaces and subsequent scanning with confocal laser-induced spectroscopy. [1,2,5,[11][12][13][14] For binding of capture molecules, different techniques for immobilization onto solid surfaces have been developed. [15,16] Several silanization techniques are known, such as the use of amino-, aldehyde-or epoxysilane films on glass and the coupling of proteins or DNA via mono-or bifunctional spacers, for example, glutardialdehyde.…”

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confidence: 99%

“…[2,13,29,[33][34][35] We prepared a nonamphiphilic "hairy-rod" cellulose architecture with terminal biotin groups. [36,37] Biotin-TMSC 4 was prepared by nucleophilic substitution of activated imidazolylcarbamatetrimethylsilylethercellulose (IDC-TMSC) intermediate 3 with a 18.9 D spacer arm of 5-(biotinamido)pentylamine (Scheme 1).…”

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confidence: 99%

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Biotin‐Functionalized Cellulose‐Based Monolayers as Sensitive Interfaces for the Detection of Single Molecules

Jung

Angerer²,

Löscher³

et al. 2006

ChemBioChem

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Ultrathin Cellulose-Based Layers for Detection of Single Antigen Molecules

Löscher¹,

Ruckstuhl

Seeger

1998

Adv. Mater.

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Use of ªlivingº catalysts for the polymerization of chiral isocyanides, in which the chiral center was relatively close to the isocyanide group, was shown to pass on the helical sense of the backbone in the polymer to achiral monomeric units, see: F. Takei, K. Yanai, K. Onitsuka, S. Takahashi, Angew. Chem. Int. Ed. Engl. 1996, 35, 1554 [15] It is not clear whether the observed variance of optical activity with ee of the monomers is a result of the formation of pure M helices from monomeric units with only R configuration, and pure P helices with S monomers (i.e., isotactic polymer), or whether the macromolecules contain unequal mixtures of R and S monomeric units and helix reversals. [16] T. [18] When poly(isocyanovalerate) was prepared under the same conditions as those described for the copolymerizations here, it had [a] 25 546 = ±67 deg cm 2 g ±1 ). Assuming that [a] 25 546 of 4 is given by: [a] 25 546 (4) = X PR [a] 25 546 (PR) + X iV [a] 25 546 (iV), where X PR and [a] 25 546 (PR) are the mole fraction and specific optical rotation resulting from the promesogenic units, respectively, and X iV and [a] 25 546 (iV) are the mole fraction and specific optical rotation of the isovalerate units, respectively, we can estimate [a] 25 546 (PR) for the copolymer 4B (which contains 20 % iminovalerate moieties), which amounts to the maximum optical rotation achievable for the poly(isocyanide), to be ±618 deg cm 2 g ±1 . (This value is comparable with the maximum optical rotation observed in an aromatic isocyanide, which is ±618 deg cm 2 g ±1 , reported by Nolte and coworkers, see [8].) The homopolymer (S)-2B is comprised of contributions from the helices of opposite sense, with equal and opposite optical rotations, such that [a] 25 546 ((S)-2B) = ±564 deg cm 2 g ±1 = X P [a] 25 546 (PR) ± X M [a] 25 546 (PR), where X P and X M are the proportions of the M and P helices, respectively. From this expression, and bearing the assumptions stated in mind, we calculate an approximate value for the diastereomeric excess of the P helix in (S)-2B of 91 %. Using the same reasoning, the diastereomeric excess of the P helix for (S)-2A is approximately 24 %.

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Counting of Single Protein Molecules at Interfaces and Application of This Technique in Early-Stage Diagnosis

Cited by 33 publications

References 37 publications

Evaluation of Confocal Fluorescence Spectroscopy for the Detection of Pathological Prion Proteins

Evaluation of Confocal Fluorescence Spectroscopy for the Detection of Pathological Prion Proteins

Biotin‐Functionalized Cellulose‐Based Monolayers as Sensitive Interfaces for the Detection of Single Molecules

Ultrathin Cellulose-Based Layers for Detection of Single Antigen Molecules

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