Ultrasensitive Fluorescence Research Articles

Multiplex microRNA Profiling from Crude Biofluids

Genetic Engineering & Biotechnology NewsVol. 35, No. 12 Drug Discovery TutorialMultiplex microRNA Profiling from Crude BiofluidsParticle-Based Approach Profiles Up to 68 miRNAs from Plasma, Serum, and ExosomesMichael Tackett, Graeme Doran, and Daniel PregibonMichael TackettMichael Tackett is director, assays services, Graeme Doran is director, genomic assays, and Daniel Pregibon (E-mail Address: dpregibon@abcam.com) is GM, platform innovation at Abcam. Website: www.abcam.com/miRNA-profiling.Search for more papers by this author, Graeme DoranMichael Tackett is director, assays services, Graeme Doran is director, genomic assays, and Daniel Pregibon (E-mail Address: dpregibon@abcam.com) is GM, platform innovation at Abcam. Website: www.abcam.com/miRNA-profiling.Search for more papers by this author, and Daniel PregibonMichael Tackett is director, assays services, Graeme Doran is director, genomic assays, and Daniel Pregibon (E-mail Address: dpregibon@abcam.com) is GM, platform innovation at Abcam. Website: www.abcam.com/miRNA-profiling.Search for more papers by this authorPublished Online:11 Jun 2015https://doi.org/10.1089/gen.35.12.08AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited bySupramolecular Microgels with Molecular Beacons at the Interface for Ultrasensitive, Amplification-Free, and SNP-Selective miRNA Fluorescence Detection25 April 2019 | ACS Applied Materials & Interfaces, Vol. 11, No. 19 Volume 35Issue 12Jun 2015 Information© 2015 by GEN PublishingTo cite this article:Michael Tackett, Graeme Doran, and Daniel Pregibon.Multiplex microRNA Profiling from Crude Biofluids.Genetic Engineering & Biotechnology News.Jun 2015.14-15.http://doi.org/10.1089/gen.35.12.08Published in Volume: 35 Issue 12: June 11, 2015PDF download

Partitioning Variability of a Compartmentalized In Vitro Transcriptional Thresholding Circuit.

Encapsulation of in vitro biochemical reaction circuits into small, cell-sized compartments can result in considerable variations in the dynamical properties of the circuits. As a model system, we here investigate a simple in vitro transcriptional reaction circuit, which generates an ultrasensitive fluorescence response when the concentration of an RNA transcript reaches a preset threshold. The reaction circuit is compartmentalized into spherical water-in-oil microemulsion droplets, and the reaction progress is monitored by fluorescence microscopy. A quantitative statistical analysis of thousands of individual droplets ranging in size from a few up to 20 μm reveals a strong variability in effective RNA production rates, which by computational modeling is traced back to a larger-than-Poisson variability in RNAP activities in the droplets. The noise level in terms of the noise strength (the Fano factor) is strongly dependent on the ratio between transcription templates and polymerases, and increases for higher template concentrations.

Electrostatic nucleic acid nanoassembly enables hybridization chain reaction in living cells for ultrasensitive mRNA imaging.

Efficient approaches for intracellular delivery of nucleic acid reagents to achieve sensitive detection and regulation of gene and protein expressions are essential for chemistry and biology. We develop a novel electrostatic DNA nanoassembly that, for the first time, realizes hybridization chain reaction (HCR), a target-initiated alternating hybridization reaction between two hairpin probes, for signal amplification in living cells. The DNA nanoassembly has a designed structure with a core gold nanoparticle, a cationic peptide interlayer, and an electrostatically assembled outer layer of fluorophore-labeled hairpin DNA probes. It is shown to have high efficiency for cellular delivery of DNA probes via a unique endocytosis-independent mechanism that confers a significant advantage of overcoming endosomal entrapment. Moreover, electrostatic assembly of DNA probes enables target-initialized release of the probes from the nanoassembly via HCR. This intracellular HCR offers efficient signal amplification and enables ultrasensitive fluorescence activation imaging of mRNA expression with a picomolar detection limit. The results imply that the developed nanoassembly may provide an invaluable platform in low-abundance biomarker discovery and regulation for cell biology and theranostics.

Ultrasensitive Fluorescence Immunoassay for Detection of Bisphenol A in Milk Products Using Functionalized Gold Nanoparticles as Probe

An ultrasensitive fluorescence immunoassay was developed for detection of bisphenol A (BPA). Herein, thiol-modified single-strand DNA (SH-ssDNAs) and anti-BPA antibodies were simultaneously conjugated with gold nanoparticles (AuNPs), and the fluorescein isothiocyanate-labeled single-strand DNA was hybridized with SH-ssDNAs to form a dual-codified probe. In the presence of BPA, a competitive immunoreaction was conducted between BPA and BPA coating antigens immobilized on microplate for the anti-BPA antibodies of dual-codified probe. Then, in order to avoid the fluorescent signals quenched by AuNPs, restriction enzyme BamH І was employed to cut the fluorescent labels from immunocomplex, resulting in an increase of fluorescence emission. Under optimized conditions, the linear range for detection of BPA is from 1.0 × 10−2 to 1.0 × 103 ng/L with a low limit of detection of 3.4 × 10−3 ng/L. This method is of good sensibility, stability, and specificity, and it can be used to detect BPA in actual milk products and its migration from packaging materials into milk.

Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low-comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls-these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.

Ultra-sensitive nanofiber fluorescence detection in a microfluidic chip.

We report an ultra-sensitive and robust fluorescence sensor made by using a biconical taper with a waist diameter of 720 nm for both excitation and fluorescence collection. To enhance the stability of the fluorescence sensor, the biconical taper has been embedded in a 125 µm wide microchannel with a detection length of 2.5 cm. Investigated by measuring the fluorescence intensity of rhodamine 6G (R6G), the sensor shows a detection limit down to 100 pM, with excellent reversibility in a concentration range of 0–10 nM. The sensor has also been applied to quantum dot (QD)-labeled streptavidin measurements, yielding a detection sensitivity down to 10 pM for QDs. In addition, the small sample volume (ca. 500 nL), high sampling throughput, and seamless connection between the biconical taper and standard optical fibers offer a number of attractive advantages for chemical and biosensing applications.

Ultrasensitive atomic fluorescence spectrometric detection of DNA with quantum dot-assemblies as signal amplification labels

Using self-assembled CdS QDs as signal amplification labels, an ultrasensitive AFS method was bridged to DNA detection, with an impressive limit of detection of 0.8 aM.

Simple and convenient G-quadruplex-based fluorescence assay of micrococcal nuclease activity

An ultrasensitive and straightforward fluorescence sensing platform for MNase activity has been developed based on NMM/G-quadruplexes.

Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots.

We demonstrate ultrasensitive fluorescence imaging of proteins on dot blots and western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CNPPV). We achieved a detection limit at the single-picogram level in dot blots; with conventional western blot, we detected 50 pg of transferrin and trypsin inhibitor after SDS-PAGE and transfer onto a PVDF membrane. In this protocol, we describe the preparation of Pdots and its streptavidin bioconjugate and the procedures of dot blot and western blots.

Detection and Quantification of Heme and Chlorophyll Precursors Using a High Performance Liquid Chromatography (HPLC) System Equipped with Two Fluorescence Detectors

[Abstract] Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and di-vinyl forms of (proto) chlorophyllide, using just a few millilitres of the cyanobacterial culture. Extracted precursors are separated by High Performance Liquid Chromatography system (HPLC) and detected by two ultra-sensitive fluorescence detectors set to different wavelengths.

Photoinduced electron transfer of poly(o-phenylenediamine)–Rhodamine B copolymer dots: application in ultrasensitive detection of nitrite in vivo

An ultrasensitive fluorescence sensing system for NO2−in vivo based on photoinduced electron transfer of poly(o-phenylenediamine)–Rhodamine B copolymer dots is built.

Ultrasensitive turn-on fluorescent detection of trace thiocyanate based on fluorescence resonance energy transfer

Thiocyanate (SCN−) is a small anion byproduct of cyanide metabolism. Several methods have been reported to measure SCN− above the micromolar level. However, SCN− is derived from many sources such as cigarettes, waste water, food and even car exhaust and its effect is cumulative, which makes it necessary to develop methods for the detection of trace SCN−. In this paper, a simple and ultrasensitive turn-on fluorescence assay of trace SCN− is established based on the fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and fluorescein. The detection limit is 0.09nM, to the best of our knowledge, which has been the lowest detection LOD ever without the aid of costly instrumentation. The fluorescence of fluorescein is significantly quenched when it is attached to the surface of AuNPs. Upon the addition of SCN−, the fluorescence is turned on due to the competition action between SCN− and fluorescein towards the surface of AuNPs. Under an optimum pH, AuNPs size and concentration, incubation time, the fluorescence enhancement efficiency [(IF−I0)/I0] displays a linear relationship with the concentration of SCN− in the range of 1.0nM to 40.0nM. The fluorescein–AuNP sensor shows absolutely high selectivity toward SCN− than other 16 anions. The common metal ions, amino acids and sugars have no obvious interference effects. The accuracy and precision were evaluated based on the recovery experiments. The cost effective sensing system is successfully applied for the determination of SCN− in milk products and saliva samples.

Ultrasensitive time-resolved microplate fluorescence immunoassay for bisphenol A using a system composed on gold nanoparticles and a europium(III)-labeled streptavidin tracer

We demonstrate an ultrasensitive time-resolved fluorescence immunoassay for bisphenol A (BPA) where gold nanoparticles (Au-NPs) were modified with anti-BPA antibody and thiolated dsDNA-biotin acting as a signal amplifier. In a competitive reaction, the analyte (BPA) competes with immobilized BPA-ovalbumin conjugate on the surface of microtiter plates to bind to the anti-BPA antibodies on the surface of the Au-NPs. In the next step, a Eu(III)-labeled streptavidin is added to link to the SH-dsDNA-biotin as a tracer. Fluorescence is amplified via both the Au-NPs and the biotin-streptavidin systems, and its intensity is measured in a time-resolved fluorescence immunoassay. The linear range is from 1.0 fg∙mL−1 to 1.0 ng∙mL−1, and the detection limit is 0.3 fg mL−1, respectively. The method was applied to the determination of BPA in spiked water sample, with recoveries ranging from 90.2 to 106.4 %.

Quaternary ammonium promoted ultra selective and sensitive fluorescence detection of fluoride ion in water and living cells.

Highly selective and sensitive fluorescent probes with a quaternary ammonium moiety have been rationally designed and developed for fast and sensitive fluorescence detection of fluoride ion (F(-) from NaF, not TBAF) in aqueous solution and living cells. With the sequestration effect of quaternary ammonium, the detection time was less than 2 min and the detection limit of fluoride ion was as low as 0.57 ppm that is among the lowest detection limits in aqueous solutions of many fluoride fluorescence probes in the literature.

Ultrasensitive fluorescence polarization aptasensors based on exonuclease signal amplification and polystyrene nanoparticle amplification.

Here, we combine T7 exonuclease (T7 Exo) signal amplification and polystyrene nanoparticle (PS NP) amplification to develop novel fluorescence polarization (FP) aptasensors. The binding of a target/open aptamer hairpin complex or a target/single-stranded aptamer complex to dye-labeled DNA bound to PS NPs, or the self-assembly of two aptamer subunits (one of them labeled with a dye) into a target/aptamer complex on PS NPs leads to the cyclic T7 Exo-catalyzed digestion of the dye-labeled DNA or the dye-labeled aptamer subunit. This results in a substantial decrease in the FP value for the amplified sensing process. Our newly developed aptasensors exhibit a sensitivity five orders of magnitude higher than that of traditional homogeneous aptasensors and a high specificity for the target molecules. These distinct advantages of our proposed assay protocol make it a generic platform for the design of amplified aptasensors for ultrasensitive detection of various target molecules.

Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays

In this work, two different configurations for novel amplified fluorescence polarization (FP) aptasensors based on nicking enzyme signal amplification (NESA) and graphene oxide (GO) enhancement have been developed for ultrasensitive and selective detection of biomolecules in homogeneous solution. One approach involves the aptamer-target binding induced the stable hybridization between an aptamer probe and a fluorophore-labeled DNA probe linked to GO, and forms a nicking site-containing duplex DNA region due to the enhancement of base stacking. The second analytical method involves the target induced the assembly of two aptamer subunits into an aptamer-target complex, and then hybridizes with a fluorophore-labeled DNA probe linked to GO, forming a nicking site-containing duplex DNA region. The formation of the duplex DNA region in both methods triggers the NESA process, resulting in the release of many short DNA fragments carrying the fluorophore from GO, generating a significant decrease of the FP value that provides the readout signal for the amplified sensing process. By using the NESA coupled GO enhancement path, the sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude over traditional aptamer-based homogeneous assays. Moreover, these aptasensors also exhibit high specificity for target molecules, which are capable of detecting target molecule in biological samples. Considering these qualities, the proposed FP aptasensors based NESA and GO enhancement can be expected to provide an ultrasensitive platform for amplified analysis of target molecules.

Identification of primary amines in Titan tholins using microchip nonaqueous capillary electrophoresis

Titan, the moon of Saturn with a thick atmosphere and an active hydrocarbon-based weather cycle, is considered the best target in the solar system for the study of organic chemistry on a planetary scale. Microfluidic devices that employ liquid phase techniques such as capillary electrophoresis with ultrasensitive laser-induced fluorescence detection offer a unique solution for in situ analysis of complex organics on Titan. We previously reported a protocol for nonaqueous microfluidic analysis of primary aliphatic amines in ethanol, and demonstrated separations of short- and long-chain amines down to −20 °C. We have optimized this protocol further, and used it to analyze Titan aerosol analogues (tholins) generated in two separate laboratories under a variety of different conditions. Ethylamine was a major product in all samples, though significant differences in amine content were observed, in particular for long-chain amines (C12–C27). This work validates microfluidic chemical analysis of complex organics with relevance to Titan, and represents a significant first step in understanding tholin composition via targeted functional group analysis.

Ultrasensitive quantum dot fluorescence quenching assay for selective detection of mercury ions in drinking water.

Mercury is one of the most acutely toxic substances at trace level to human health and living thing. Developing a rapid, cheap and water soluble metal sensor for detecting mercury ions at ppb level remains a challenge. Herein, a metal sensor consisting of MPA coated Mn doped ZnSe/ZnS colloidal nanoparticles was utilized to ultrasensitively and selectively detect Hg2+ ions with a low detection limit (0.1 nM) over a dynamic range from 0 to 20 nM. According to strong interaction between thiol(s) and mercury ions, mercaptopropionic acid (MPA) was used as a highly unique acceptor for mercury ions in the as-obtained ultrasensitive sensor. In the presence of mercury ions, colloidal nanoparticles rapidly agglomerated due to changes of surface chemical properties, which results in severe quenching of fluorescent intensity. Meanwhile, we find that the original ligands are separated from the surface of colloidal nanoparticles involving strongly chelation between mercury ion and thiol(s) proved by controlled IR analysis. The result shows that the QD-based metal ions sensor possesses satisfactory precision, high sensitivity and selectivity, and could be applied for the quantification analysis of real samples.

A novel ultrasensitive carboxymethyl chitosan-quantum dot-based fluorescence “turn on–off” nanosensor for lysozyme detection

In this work, we developed an ultrasensitive “turn on–off” fluorescence nanosensor for lysozyme (Lyz) detection. The novel nanosensor was constructed with the carboxymethyl chitosan modified CdTe quantum dots (CMCS-QDs). Firstly, the CMCS-QDs were fabricated via the electrostatic interaction between amino groups in CMCS polymeric chains and carboxyl groups on the surface of QDs. In the fluorescence “turn-on” step, the strong binding ability between Zn2+ and CMCS on the surface of QDs can enhance the photoluminescence intensity (PL) of QDs. In the following fluorescence “turn-off” step, the N-acetyl-glucosamine (NAG) section along the CMCS chains was hydrolyzed by Lyz. As a result, Zn2+ was released from the surface of QDs, and the Lyz–QDs complexes were formed to quench the QDs PL. Under the optimal conditions, there was a good linear relationship between the PL of QDs and the Lyz concentration (0.1–1.2ng/mL) with the detection limit of 0.031ng/mL. The developed method was ultrasensitive, highly selective and fast. It has been successfully employed in the detection of Lyz in the serum with satisfactory results.

Highly photostable “super”-photoacids for ultrasensitive fluorescence spectroscopy

The photoacid 8-hydroxypyren-1,3,6-trisulfonic acid (HPTS, pyranine) is a widely used model compound for the examination of excited state proton transfer (ESPT). We synthesized five "super"-photoacids with varying hydrophilicity and acidity on the basis of HPTS. By chemical modification of the three sulfonic acid substituents, the photoacidity is enhanced by up to more than five logarithmic units from pK*≈ 1.4 to ∼-3.9 for the most acidic compound. As a result, nearly quantitative ESPT in DMSO can be observed. The novel photoacids were characterized by steady-state and time-resolved fluorescence techniques showing distinctively red shifted spectra compared to HPTS while maintaining a high quantum yield near 90%. Photostability of the compounds was checked by fluorescence correlation spectroscopy (FCS) and was found to be adequately high for ultrasensitive fluorescence spectroscopy. The described photoacids present a valuable palette for a wide range of applications, especially when the properties of HPTS, i.e. highly charged, low photostability and only moderate excited state acidity, are limiting.