Highly efficient single molecule detection in microstructures

Dörre, Klaus; Stephan, Jens; Lapczyna, Markus; Stuke, M.; Dunkel, Holger; Eigen, Manfred

doi:10.1016/s0168-1656(00)00415-6

Cited by 27 publications

(14 citation statements)

References 29 publications

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“…In addition, the linear dependence of transit time on flow rate was observed (data not shown) as determined from the autocorrelation function (see Figure 2e) and also, the number of photon burst events as a function of particle concentration and particle velocity (see Figures S1 and S2 in the Supporting Information). 33…”

Section: Resultsmentioning

confidence: 99%

Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity

Lee

et al. 2010

Anal. Chem.

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We have developed an instrument for spectral cross-talk free dual-color fluorescence crosscorrelation spectroscopy (FCCS), which provides a readout modality for the study of enzyme activity in application areas such as high throughput screening. Two spectrally distinct (∼250 nm) fluorophores, Cy3 and IRD800, were excited simultaneously using two different excitation sources; one poised at 532 nm and the other at 780 nm. The fluorescence information was processed on two different color channels monitored with single photon avalanche diodes (SPADs) that could transduce events at the single-molecule level. The system provided no color cross-talk (crossexcitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET), significantly improving data quality. To provide evidence of cross-talk free operation, the system was evaluated using bright microspheres (λ abs = 532 nm, λ em = 560 nm) and quantum dots (λ abs = 532 nm, λ em = 810 nm). Experimental results indicated that no color leakage from the microspheres or quantum dots into inappropriate color channels was observed. To demonstrate the utility of the system, the enzymatic activity of APE1, which is responsible for nicking the phosphodiester backbone in DNA on the 5′ side of an apurinic/apyrimidinic site, was monitored by FCCS using a double-stranded DNA substrate dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid) of the enzyme using this cross-talk free FCCS platform. In all cases, no spectral leakage from single molecule events into inappropriate color channels was observed.

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Section: Resultsmentioning

confidence: 99%

Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity

Lee

et al. 2010

Anal. Chem.

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“…All buffers and solvents were purified prior to use as commercially available solutions show significant concen-trations of fluorescent impurities. Inorganic buffers have to be used in order to be able to remove organic substances efficiently by activated carbon [37].…”

Section: Dyes and Buffersmentioning

confidence: 99%

“…In the case of efficient and nearly complete single molecule detection signals are recorded that do not originate from the probe, but are a result of impurities from the immersion liquid. Especially in the green/yellow spectral region (480-600 nm) this problem causes serious difficulties [37,51]. For excitation wavelengths above 620 nm the problem of contaminations becomes less significant as most biological probes do not show sufficient single molecule fluorescence in this spectral range [35].…”

Section: Fig 4 Normalized Autocorrelation Curve For Tmr In Water Inmentioning

confidence: 99%

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Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Dörre¹,

Stephan²,

Eigen

2001

Single Mol.

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In this article we describe a detailed comparison between single molecule detection in three different types of microchannels. The overall detection efficiencies and the signal-to-noise ratios are compared. A new data evaluation technique that is used is explained in detail. The average number of photons per molecule is calculated from the measurements and the detection properties of each channel with respect to the diffusional characteristics of the dye molecules are investigated. From the detection efficiencies advantages and drawbacks of the individual methods are elucidated as well as possible applications of the techniques are illustrated.

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“…Single molecules have been detected by means of fluorescence in relatively large capillaries, 4-6 micrometer 7-10 and even submicrometer sized channels. [11][12][13][14][15] Previous work in our laboratories reported on the detection of flowing fluorescent particles like bacteria and microspheres in a microcapillary mounted on a confocal fluorescence microscopy setup. 16 These results led to the design of a microfluidic biochip aimed at detection and sorting of relatively small particles in real time using the same confocal fluorescence microscope.…”

Section: Introductionmentioning

confidence: 99%

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

Kunst

Schots

Visser

2004

Review of Scientific Instruments

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An instrumental system is described for detecting and sorting single fluorescent particles such as microspheres, bacteria, viruses, or even smaller macromolecules in a flowing liquid. The system consists of microfluidic chips (biochips), computer controlled high voltage power supplies, and a fluorescence microscope with confocal optics. The confocal observation volume and detection electro-optics allow measurements of single flowing fluorescent particles. The output of the avalanche photodiode (single photon detector) is coupled to a real-time photon-burst detection device, which output can address the control of high voltage power supplies for sorting purposes. Liquid propulsion systems like electro-osmotic flow and plain electric fields to direct the particles through the observation volume have been tested and evaluated. The detection and real-time sorting of fluorescent microspheres are demonstrated. Applications of these biochips for screening of bacteriophages-type biolibraries are briefly discussed.

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Highly efficient single molecule detection in microstructures

Cited by 27 publications

References 29 publications

Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity

Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity

Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

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