Sean M. Burrows

Detection of miRNA using a double-strand displacement biosensor with a self-complementary fluorescent reporter.

Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

Mapping vortex-like hydrodynamic flow in microfluidic networks using fluorescence correlation spectroscopy

The ability to quickly measure flow parameters in microfluidic devices is critical for micro total analysis system (microTAS) applications. Macrofluidic methods to assess flow suffer from limitations that have made conventional methods unsuitable for the flow behavior profiling. Single molecule fluorescence correlation spectroscopy (FCS) has been employed in our study to characterize the fluidic vortex generating at a T-shape junction of microscale channels. Due to its high spatial and temporal resolution, the corresponding magnitudes relative to different flow rates in the main channel can be quantitatively differentiated using flow time (tau(F)) measurements of dye molecules traversing the detection volume in buffer solution. Despite the parabolic flow in the channel upstream, a heterogeneous distribution of flow has been detected across the channel intersection. In addition, our current observations also confirmed the aspect of vortex-shaped flow in low-shear design that was developed previously for cell culture. This approach not only overcomes many technical barriers for examining hydrodynamic vortices and movements in miniature structures without physically integrating any probes, but it is also especially useful for the hydrodynamic studies in polymer-glass based micro-reactor and -mixer.

Light Tolerance of R-Phycoerythrin and a Tandem Conjugate Observed by Single Molecule Recrossing Events

The recrossing of single molecules in a probe volume was used to investigate light harvesting and energy transfer between R-phycoerythrin (R-PE) and a tandem conjugate dye. The normalized recrossing ratio, Nr/Nt, was defined as the number of molecules that reenter the probe volume (Nr) to the total number of molecules detected (Nt). The energy transfer process in phycobiliproteins was studied as a function of excitation irradiation and irradiation time. This was achieved by investigating the average baseline-subtracted fluorescence intensity, normalized molecular recrossing ratio (Nr/Nt), and the number of molecules detected per second. The photon saturation irradiance of the R-PE and the tandem conjugate were compared with each other, showing that energy transfer to the tandem dye significantly improves photostability and light tolerance of the phycobiliprotein. The Nr/Nt ratio was used to study the photophysical properties of R-phycoerythrin and the tandem conjugate Streptavidin R-Phycoerythrin-AlexaFluor-647 (PE-647). Normalized molecular recrossings showed that energy transfer to a tandem conjugate could reduce the formation of triplet states in R-phycoerythrin and extend the light tolerance of certain phycobiliproteins.

Fluorescent microRNA biosensors: a comparison of signal generation to quenching

Many microRNA biosensor platforms use fluorescence signal generation or quenching; however, signal generation is often regarded as the superior method. An argument can be made that if the noise is the same for both methods, then there should be no difference between the two methods. Current literature details the analytical figures of merit (FOM) for transduction and recognition mechanisms that use either signal generation or quenching, but lacks a direct comparison using the same fluorescent reporter molecule. Here we provide such a direct comparison. The signal-on and signal-off fluorescence metrics were found to be comparable rather than competitive. We found fluorescence enhancement provides marginal improvements to sensitivity and limits of detection (LOD) over fluorescence quenching. In fact, both transduction mechanisms are capable of picomolar LOD. The role thermodynamics plays on the sensitivity and LOD are discussed. Both signal-on and signal-off gave statistically similar signal-to-noise ratios. Finally, the selectivity of the two recognition mechanisms for miRNA detection will be addressed. In the future, we will use this knowledge to advance highly sensitive and selective in situ microRNA sensors for cell and tissue imaging.

Förster resonance energy transfer to impart signal-on and -off capabilities in a single microRNA biosensor

Many studies have found that over- or under-expression of biomolecules called microRNAs (miRNA) regulates several diseases. Biosensors are in need to visually identify the relative expression level of miRNA to determine the direction these miRNA change in cells and tissues. Our established reporter+probe miRNA biosensor design requires that miRNA outcompete and displace the reporter from the probe. Once displaced, the reporter folds into a hairpin structure to force together a pair of Förster Resonance Energy Transfer (FRET) dyes. The donor and acceptor signal changes can be used to indicate the over-/under-expression of miRNA. The bright signal from the donor will indicate miRNA under-expression; the bright acceptor signal will indicate miRNA over-expression. Since close proximity of the dyes to each other and nucleic acids often quench fluorescence, polyethylene glycol spacers were added in-between the dyes and nucleic acids. We compared reporter designs with and without spacers to investigate the effects on the following analytical metrics: (1) extent of signal change, (2) limits of detection and quantitation, and (3) sensitivity. Systematic errors and amount of reporter+probe biosensor formed were evaluated for one of the biosensors. Cy3|Cy5 and 6-carboxyfluorescein (6-FAM)|ATTO 633 dye pairs on reporters containing spacers showed an increase in the acceptor signal change by ∼190 and ∼484%, respectively, compared to no spacers. Transduction mechanisms that enhance and quench the signal both showed LODs that ranged from 3-17 nanomolar (nM) with 100 nM of the biosensor.

Achieving plasmon reproducibility from surfactant free gold nanostar synthesis

Obtaining reproducible plasmon resonances from nanostars remains a challenge for both surfactant and surfactant-free syntheses. For any nanostar application, a plasmon band with a reproducible spectral profile and λmax is a fundamental criterion. In particular, synthesis of biocompatible gold nanostars will benefit from surfactant-free methods to alleviate concerns over the cytotoxicity of many surfactants used in current synthesis techniques and the relative ease of synthesis. In this paper, we analyze different surfactant-free nanostar synthesis conditions and their influence on achieving plasmon reproducibility. Plasmon reproducibility was judged via the standard deviation of the extinction spectras λmax and the spectral bandwidth. The synthesis temperature was the most influential factor in producing gold nanostars with reproducible plasmons. Nanostars synthesized at 5 °C exhibited a statistically (α = 0.05) smaller standard deviation in both their λmax and spectral bandwidth than nanostars synthesized at 25 °C. The reproducibility of the plasmon band was preserved even when the reaction conditions were adjusted to shift the position of the peak plasmon resonance. The high reproducibility of this approach, combined with the ease of synthesis, presents a significant step towards achieving gold nanostars with reproducible plasmons for biological applications. For example, photodynamic therapy, biomedical imaging contrast agents, and biosensing will all benefit from the reproducibility of the nanostars plasmon bands.

Molecular Approaches To Address the Challenges of RNA Analysis in Complex Matrices

We present on a design change and addition of an internal polyethylene glycol (PEG) spacer to an existing biosensor. There were two reasons for changing the sensor design. The first was to increase the stability of the biosensor to avoid binding off-analytes with single nucleotide polymorphisms. The second was to prevent sensor degradation by nucleases. The biosensor, designed for detection of short noncoding RNA strands, is composed of Reporter and Probe nucleic acid strands that form a partially complementary duplex. The internal PEG was added to the Reporter, and subsequently diminished false negatives that resulted from off-oligonucleotide binding. Furthermore, the PEG eliminated degradation of the sensor by DNase1 endonuclease. Currently, in situ and crude cell lysate RNA analysis is hindered by nonspecific interactions and degradation by endogenous nucleases. Together, the design changes presented here mitigate these matrix effects and allow for robust RNA analysis in complex media.

Dye-Specific Wavelength Offsets to Resolve Spectrally Overlapping and Co-Localized Two-Photon Induced Fluorescence

The fundamentally important fluorescent imaging has one major limitation, resolution of over three dyes. This limitation is in part due to the overlap of the broad emission profile of each of the emitters used in fluorescence detection. The overlapping emission contaminates each emitters detection channel, referred to as cross-talk. To reduce fluorescence cross-talk for two photon applications, we present an innovative Two-Photon-Dye-Specific Excitation-Emission Offset (TP-DSO) method. TP-DSO selectively detects each dye by synchronously scanning the excitation and emission wavelengths at defined wavelength offsets. This technique advances multicolor analysis significantly by resolving dyes with highly overlapping spectral profiles. We identified three benefits: reduced excitation spectral bandwidth, reduced emission cross-talk between colocalized emitters with closely overlapping fluorescence, and validated use of thin-film variable optical emission filters for tuning the bandwidth and center wavelength. TP-DSO will advance multicolor analysis for many applications.