Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes

Hsieh, Albert Tsung-Hsi; Pan, Patrick Jen-Hao; Lee, Abraham P.

doi:10.1007/s10404-009-0406-9

Cited by 51 publications

(37 citation statements)

References 57 publications

(53 reference statements)

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“…These FRET methods are suitable for integration with microfluidic devices for on-chip detection. For example, molecular beacon probes are a well-known FRET technology and have been combined with a microfluidic picoliter drop generator for the rapid detection of target DNA sequences [8]. Molecular beacons comprise a DNA hairpin (also called a stem-loop structure) labeled at opposite termini with donor and acceptor dyes (Fig.…”

Section: Bioassaysmentioning

confidence: 99%

“…7b). Hybridization was complete within 10 s. BRCA1 could be detected at concentrations between 125 nM and 2 mM with 2 mM molecular beacon, or at concentrations as low as 500 fM with 2.5 nM molecular beacon [8]. The recovery of FRET-quenched fluorescence in the microfluidic channels was visualized using a cooled monochrome CCD camera with filter sets for Cy3 and fluorescein.…”

Section: Bioassaysmentioning

confidence: 99%

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Fluorescence Resonance Energy Transfer (FRET)

Algar

2013

Encyclopedia of Microfluidics and Nanofluidics

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SynonymsElectronic energy transfer (EET); Förster resonance energy transfer (FRET) Definition Fluorescence resonance energy transfer (FRET) is a non-radiative, through-space excitation energy transfer process. The electronic excitation energy of a donor molecule is transferred to a ground-state acceptor through dipole-dipole interactions without involvement of a photon or molecular contact. Efficient FRET requires close proximity and suitable alignment between the transition dipoles of the donor and acceptor, as well as overlap between the donor emission spectrum and acceptor absorption spectrum. OverviewFRET was first elucidated by Theodor Förster between 1946 and 1948 [1]. Many researchers, including the International Union of Pure and Applied Chemistry (IUPAC), recognize FRET as Förster resonance energy transfer rather than fluorescence resonance energy transfer. The tribute to Förster notwithstanding, it is argued that the "fluorescence" terminology is incorrect because no photon is emitted during the energy transfer process. However, there is some value to the fluorescence resonance energy transfer terminology, which can be used to distinguish between energy transfer from optically excited donors and energy transfer from excited-state donors formed as the products of chemical or enzyme-catalyzed reactions. The latter two processes also follow the mechanism elucidated by Förster and are commonly referred to as chemiluminescence resonance energy transfer (CRET) and bioluminescence resonance energy transfer (BRET), respectively. In practice, both the "Förster" and "fluorescence" terminology are widely accepted expansions of the FRET acronym. Despite Förster's breakthrough in the mid-twentieth century, it was not until the 1990s that improvements in fluorescence instrumentation and the availability of fluorescent materials made it possible for the full potential of FRET to be realized [2]. Currently, FRET is one of the most powerful and widely utilized fluorescence techniques, with numerous applications in biology, biochemistry, biophysics, and bioanalysis.Physically, the FRET process is summarized in Eq. 1, where D represents the donor fluorophore, A represents the acceptor chromophore, and * indicates an excited electronic state.

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Section: Bioassaysmentioning

confidence: 99%

Section: Bioassaysmentioning

confidence: 99%

Fluorescence Resonance Energy Transfer (FRET)

Algar

2013

Encyclopedia of Microfluidics and Nanofluidics

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“…In droplet-based microfluidics, micro/nanoliter-sized droplets are generated, transported, mixed, reacted, synthesized, or analyzed with the advantages of precise control of the droplet volume and reaction time, parallel operations at a high rate of throughputs, and flexible and adaptable to a variety of assays (3), (4) . Moreover, the isolated micro-environment created in the individual droplets may provide unique culture and growth space for single cell study (5) .…”

Section: Introductionmentioning

confidence: 99%

Micromixing of Fluids within Droplets Generated on Centrifugal Microfluidics

Chen

Tseng

2013

JFST

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Experiments were carried out to visualize mixing and reaction of two water-based solutions inside the droplets generated in the microchannels on a rotating disk. The microchannels were composed of a Y-junction to bring the two solutions into contact, a flow-focusing junction to form water-in-oil droplets, and a U-shaped channel to further enhance the droplet mixing. In the centrifugal microfluidics, plug-type droplets of 582-820 μm in length were formed at rotational speeds in the range 400-500 rpm. The mixing efficiency can be highly enhanced to reach as much as 85% for the lower rotational speed where smaller droplets are formed due to lower dispersed-phase flow velocity.

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“…For comparison, all the sequences are the same as those in Ref. [7], in which the conventional MB method was used and showed a very small discrimination ratio of 1.5:1 (1.2:1) between PM DNA and SM-M (SM-E) DNA. Figure S5 a in the Supporting Information shows the spectra for [DNA]/[MB] = 1:1 and clearly demonstrates that our method is capable of differentiating PM DNA and SM DNA (discrimination ratio greater than 100), regardless of the position of the mismatch.…”

mentioning

confidence: 99%

“…This result is consistent with the conventional MB method. [7] Note that although both PM DNA and SM-E DNA can lase at high concentrations, the difference in the corresponding lasing threshold still allows differentiation between PM DNA and SM DNA (SM-E and SM-M). As shown in Figure S6 in the Supporting Information, for a given DNA concentration, PM DNA has the lowest lasing threshold, whereas SM-M DNA has the highest.…”

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confidence: 99%

Distinguishing DNA by Analog‐to‐Digital‐like Conversion by Using Optofluidic Lasers

Sun

Fan

2011

Angew Chem Int Ed

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Distinguishing a target DNA from a counterpart that has a single base mismatch provides critical information for disease diagnosis, personalized medicine, and basic biochemical research. [1][2][3] In traditional, fluorescence-based detection, samples are placed in a cuvette, and a DNA probe is used to hybridize with the target DNA and generate a fluorescent signal. However, because of the small difference in the binding affinity for the DNA probe between the target and the strand with a single base mismatch, the discrimination ratio between the resulting fluorescence is almost unity, [4][5][6][7][8] which makes it difficult to directly and selectively detect the target DNA from a pool of mismatched DNA strands. [9,10] Herein, we describe a system for the highly specific intracavity detection of DNA that uses an optofluidic laser. This type of laser is an emerging technology that synergistically integrates a dye laser and microfluidics for miniaturized laser sources, easy sample delivery, and extremely small sample volumes. [11][12][13] In our detection system, DNA samples and probes are incorporated as part of the laser gain medium. Stimulated laser emission, rather than fluorescence (that is, spontaneous emission), is employed as the sensing signal to achieve conversion that is similar to analog-to-digital, which significantly amplifies the small intrinsic thermodynamic difference between the target and its single base mismatched counterpart. A perfectly matched (PM) DNA, a single base mismatched (SM) DNA, and a molecular beacon (MB) probe were used as a model system. A theoretical analysis was performed to elucidate the underlying intracavity detection principle. Then, a discrimination ratio (that is, R = I PM /I SM , where I PM and I SM is the light intensity generated by PM DNA and SM DNA, respectively) of 240:1 was achieved experimentally between PM DNA and SM DNA, which is an increase of over two orders of magnitude relative to the fluorescence-based method. The selective detection of PM DNA from a pool of SM DNA at a concentration ratio of 1:50 is presented. This system can also distinguish more complicated DNA sequences, such as a breast cancer sequence from a corresponding sequence that contains a single point mutation, in both buffer and serum.An MB is a DNA probe with a stem-loop structure and a dye as well as a quencher attached to each end of the sequence (Figure 1 a). [10,[14][15][16][17] Both PM DNA and SM DNA are able to hybridize with the MB. Consequently, a fraction of MBs open and generate fluorescence. This fluorescencebased detection (see the detailed analysis in Section I A in the Supporting Information) can be regarded as "analog" detection, in which a small thermodynamic difference between PM DNA and SM DNA results in a small difference in the fluorescence signal. Figure S1 in the Supporting Information shows an example of the fluorescence from PM DNA and SM DNA, which has a low discrimination ratio.In our intracavity DNA detection system, an optofluidic ring resonator (OFRR) is used as th...

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Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes

Cited by 51 publications

References 57 publications

Fluorescence Resonance Energy Transfer (FRET)

Fluorescence Resonance Energy Transfer (FRET)

Micromixing of Fluids within Droplets Generated on Centrifugal Microfluidics

Distinguishing DNA by Analog‐to‐Digital‐like Conversion by Using Optofluidic Lasers

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