Two-step Fluorescence Research Articles

Synchronously Sensitive Immunoassay and Efficient Inactivation of Living Zika Virus via DNAzyme Catalytic Amplification and In Situ Aggregation-Induced Emission Photosensitizer Generation.

Sensitive identification and effective inactivation of the virus are paramount for the early diagnosis and treatment of viral infections to prevent the risk of secondary transmission of viruses in the environment. Herein, we developed a novel two-step fluorescence immunoassay using antibody/streptavidin dual-labeled polystyrene nanobeads and biotin-labeled G-quadruplex/hemin DNAzymes with peroxidase-mimicking activity for sensitive quantitation and efficient inactivation of living Zika virus (ZIKV). The dual-labeled nanobeads can specifically bind ZIKV through E protein targeting and simultaneously accumulate DNAzymes, leading to the catalytic oxidation of Amplex Red indicators and generation of intensified aggregation-induced emission fluorescence signals, with a detection limit down to 66.3 PFU/mL and 100% accuracy. Furthermore, robust reactive oxygen species generated in situ by oxidized Amplex Red upon irradiation can completely kill the virus. This sensitive and efficient detection-inactivation integrated system will expand the viral diagnostic tools and reduce the risk of virus transmission in the environment.

Horseradish Peroxidase-Encapsulated Fluorescent Bio-Nanoparticle for Ultra-Sensitive and Easy Detection of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) has been a fascinating target in various chemical, biological, clinical, and industrial fields. Several types of fluorescent protein-stabilized gold nanoclusters (protein-AuNCs) have been developed for sensitive and easy detection of H2O2. However, its low sensitivity makes is difficult to measure negligible concentrations of H2O2. Therefore, to overcome this limitation, we developed a horseradish peroxidase-encapsulated fluorescent bio-nanoparticle (HEFBNP), comprising bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and horseradish peroxidase-stabilized gold nanoclusters (HRP-AuNCs). The fabricated HEFBNP can sensitively detect H2O2 owing to its two properties. The first is that HEFBNPs have a continuous two-step fluorescence quenching mechanism, which comes from the heterogenous fluorescence quenching mechanism of HRP-AuNCs and BSA-AuNCs. Second, the proximity of two protein-AuNCs in a single HEFBNP allows a reaction intermediate (•OH) to rapidly reach the adjacent protein-AuNCs. As a result, HEFBNP can improve the overall reaction event and decrease the loss of intermediate in the solution. Due to the continuous quenching mechanism and effective reaction event, a HEFBNP-based sensing system can measure very low concentrations of H2O2 up to 0.5 nM and show good selectivity. Furthermore, we design a glass-based microfluidic device to make it easier use HEFBNP, which allowed us to detect H2O2 with the naked eye. Overall, the proposed H2O2 sensing system is expected to be an easy and highly sensitive on-site detection tool in chemistry, biology, clinics, and industry fields.

FRET cascade miRNA addition probe from non-crosstalk DNA photonic wire assisted with clustering algorithm for early diagnosis of acute myocardial infarction

Early and accurate diagnosis of acute myocardial infarction (AMI) can significantly reduce patient mortality. A variety of miRNAs are found to dysregulate in AMI patients, but the up- or down-regulation of a specific miRNA may not be evident in the early stage, making it difficult to achieve accurate diagnosis. Here, proposing the design that DNA photonic wire (PW) with no spectral crosstalk would make an excellent template for miRNA conjoint analysis, we report the construction of a miRNA addition probe for the additive analysis of two up-regulated miRNAs (miR-133a and miR-208a) for early diagnosis of AMI in clinical serum samples. A three-dye non-crosstalk DNA PW is built to form the two-step fluorescence resonance energy transfer (FRET) cascade system, in which three paths can blocking the FRET cascade for separate or additive analysis of the two miRNAs. K-Means clustering algorithm is further utilized to classify the output signals of the miRNA addition probe, achieving a 100% accurate diagnosis of early AMI in both the training (n = 40) and validation (n = 19) cohorts of clinical serum samples.

Preparation and photoluminescence of functionalized cotton fabric by double luminescent guests-encapsulated ZnBDC metal-organic framework

A series of functionalized cotton fabrics (LG-ZnBDC-CCtn) were prepared by attachment of ZnBDC metal-organic framework with LbL deposition method on carboxylated cotton fabric (CCtn) and further encapsulation of ZnBDC with different luminescent guests (LG = Tb3+, La3+, Rhodanmine B or/and acridine orange). The characterization of LG-ZnBDC-CCtn by XRD, SEM and EDS showed that the structural integrity and morphology of ZnBDC on the CCtn surface were not destroyed after encapsulation. The photoluminescence behavior of sample LG-ZnBDC-CCtn was investigated by fluorescence excitation and emission spectra. After encapsulated with different luminescent guests, samples LG-ZnBDC-CCtn emitted different fluorescence. Although sample Tb/RhB-ZnBDC-CCtn with double LGs (Tb3+ and RhB) showed almost same visible color with RhB-ZnBDC-CCtn under sunlight, the orange yellow fluorescence of sample Tb/RhB-ZnBDC-CCtn was quite different with that of the single LG encapsulated sample Tb-ZnBDC-CCtn (green) or RhB-ZnBDC-CCtn (rose). A similar phenomenon was also observed in La/AO-ZnBDC-CCtn system. The fluorescence change of sample Tb/RhB-ZnBDC-CCtn could be due to a two-step fluorescence resonance energy transfer (FRET) among ZnBDC framework and luminescent guests. The visible color-change properties and technical performance of sample LG-ZnBDC-CCtn were evaluated with LAB color space and color strength.

NIR-II bioluminescence for in vivo high contrast imaging and in situ ATP-mediated metastases tracing

Bioluminescence imaging has been widely used in life sciences and biomedical applications. However, conventional bioluminescence imaging usually operates in the visible region, which hampers the high-performance in vivo optical imaging due to the strong tissue absorption and scattering. To address this challenge, here we present bioluminescence probes (BPs) with emission in the second near infrared (NIR-II) region at 1029 nm by employing bioluminescence resonance energy transfer (BRET) and two-step fluorescence resonance energy transfer (FRET) with a specially designed cyanine dye FD-1029. The biocompatible NIR-II-BPs are successfully applied to vessels and lymphatics imaging in mice, which gives ~5 times higher signal-to-noise ratios and ~1.5 times higher spatial resolution than those obtained by NIR-II fluorescence imaging and conventional bioluminescence imaging. Their capability of multiplexed imaging is also well displayed. Taking advantage of the ATP-responding character, the NIR-II-BPs are able to recognize tumor metastasis with a high tumor-to-normal tissue ratio at 83.4.

Correcting the effect of the detection angular on laser-induced chlorophyll fluorescence

Chlorophyll fluorescence has been widely used to monitor vegetation growth status and quantitative remote sensing monitoring of vegetation biochemical content. Therefore, it is significant to accurately measure the fluorescence information. In this study, the variation in fluorescence intensity of vivo vegetation leaf with the change in detection angle on the basis of laser-induced fluorescence technology was discussed. Experimental results demonstrated that the relationship between the emission fluorescence intensity and detection angles could be explained by the cosine expression. Then, two-step fluorescence intensity correction method was proposed. Firstly, the fluorescence intensity was corrected based on the changing of cosine expression. Secondly, the fluorescence ratio calculated based on the corrected fluorescence intensity. Results demonstrated that the effect of detection angles on fluorescence signals can be efficiently eliminated compared to the traditional spectral ratio method. Findings of this study may be valuable in promoting the applications of laser-induced fluorescence in remote sensing to achieve accurate chlorophyll fluorescence information for quantitative monitoring of plant nutrient stress.

Broad Spectrum Tunable Photoluminescent Material Based on Cascade Fluorescence Resonance Energy Transfer between Three Fluorophores Encapsulated within the Self-Assembled Surfactant Systems.

A broad spectrum tunable photoluminescent material with dual encryption based on a two-step fluorescence resonance energy transfer (FRET) between pyrene (Py), coumarin 480 (Cou480), and rhodamine 6G (R6G) in micelles of SDS and bmimDS is presented. The phenomenon is achievable due to the encapsulation of the fluorophores within these micelles. The transfer of energy as FRET between the pair Py and Cou480 showed ON at 336 nm and OFF at 402 nm in contrast to the FRET observed between the pair Cou480 and R6G that showed ON at 402 nm and OFF at 336 nm. However, the transfer of energy as FRET occurs from Py to R6G in the presence of Cou480 when excited at 336 nm, thereby making it a chain of three fluorophores with Cou480 acting as a relay fluorophore receiving energy from Py and transferring it to R6G. The different FRET scenarios between the three fluorophores in micelles provide a window for the generation of a matrix of colors, which occupies a significant 2D area in the chromaticity diagram, having potential applications in security printing. The different fluorophoric ratios generate different colors based on their individual photonic emissions and the FRET processes taking place between them. Writing tests were carried out using varied ratios of the fluorophores in the micellar systems producing different colored outputs under the UV light with insignificant visibility under the white light. We envision that this as-discovered three fluorophoric FRET system could form the basis for the future development of multi-FRET light-harvesting devices and anti-counterfeiting security inks based on much simpler non-covalent interaction aided encapsulation of the fluorophores within the self-assembled soft systems.

A smart single molecular probe for Cu2+/Fe3+/Mg2+ by three-emission behaviors

An azacrown[NS2O2]-azastyryl-modified fluorene probe (AC-AS-F) has been designed and synthesized. Systematic optical studies show that AC-AS-F displays a distinct two-step fluorescence response to the increasing quantity of Cu2+: The fluorescent emission of this compound centered at 490 nm is first blue-shifted to 450 nm with a concomitant increase in fluorescence intensity and then is only enhanced along with successive increase in Cu2+ amount from 0 to 1 and 1–2 equiv. In contrast, addition of Fe3+ leads to just an opposite manner in the fluorescence spectrum: the corresponding fluorescence emission is red-shift to 540 nm simultaneously accompanied by a obvious decrease in fluorescence intensity. After adding Mg2+, the maximum fluorescence emission of AC-AS-F is red-shifted to 510 nm synchronously but with a little decrease in the fluorescence intensity. Meanwhile AC-AS-F also displays three obviously distinct absorption behaviors with diverse absorption-ratiometric signals upon respective addition of Cu2+, Fe3+ or Mg2+, which induce an obvious change in the solution color from pale yellow to yellowish-brown and then to dark yellow upon successive addition of Mg2+, Cu2+ and Fe3+. These, together with the dual optical responses to the metal displacement of AC-AS-F-M(M = Cu2+/Mg2+) by Fe3+/Cu2+, endow AC-AS-F the multi-stimuli-channel sensing nature for these metal ions, in particular for Cu2+ and Fe3+ with fast-response function.

Two-photon-like microscopy with orders-of-magnitude lower illumination intensity via two-step fluorescence.

We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padron's quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. We also show two-step and two-photon imaging can be combined to give quartic non-linearity, further improving imaging in challenging samples. With further improvements, two-step fluorophores could replace conventional fluorophores for many imaging applications.

Construction of logic gates with the fluorene-based small molecule/DNA probes

Fluorene-based small molecules (FSMs) have optical properties and can interact with DNA. In this paper, the integrated "INH" and "AND" gates operating in parallel are developed with the fluorene-based small molecule (FSM)/DNA probe. They are activated by taking advantage of the two-step fluorescence resonance energy transfer (FRET) process and the sequence-recognition mechanism of DNA. Then, a "NOT" gate is obtained with a molecular beacon-like probe (FSM-MB) by using the FSM as the fluorophore. Moreover, the "NOT" gate based on the FSM-MB probe can be used as a biosensor and has potential applications in label-free detection of target molecules.

Two-step resonance energy transfer between dyes in layered silicate films

Single- and two-step fluorescence resonance energy transfer (FRET) was investigated between laser dyes rhodamine 123 (R123), rhodamine 610 (R610), and oxazine 4 (Ox4). The dye molecules played the role of molecular antennas and energy donors (ED, R123), energy acceptors (EA, Ox4), or both (R610). The dye cations were embedded in the films based on layered silicate laponite (Lap) with the thickness of several μm. Optically homogeneous films were prepared directly from dye/Lap colloids. Dye concentration in the films was high enough for FRET to occur but sufficiently low to prevent the formation of large amounts of molecular aggregates. The films were characterized by absorption and fluorescence spectroscopies, and their optical properties were compared with colloid precursors and dye aqueous solutions. The phenomenon of FRET was confirmed by means of steady-state and time-resolved fluorescence spectroscopies. Significant quenching of ED emission in favor of the luminescence from EA molecules was observed. FRET led to the decrease in the lifetimes of excited states of ED molecules. Molecular orientation of dye molecules was determined by polarized absorption and fluorescence spectroscopies. Almost parallel orientation with respect to silicate surface (∼30°) was determined for all fluorescent species of the dyes. Theoretical model on relationship between anisotropy and molecular orientation of the fluorophores fits well with measured data. The analysis of anisotropy measurements confirmed the significant role of FRET in the phenomenon of light depolarization.

Fluorescence detection of mercury(II) and lead(II) ions using aptamer/reporter conjugates

We have developed a fluorescence technique for the detection of Hg2+ and Pb2+ ions using polythymine (T33)/benzothiazolium-4-quinolinium dimer derivative (TOTO-3) and polyguanine (G33)/terbium ions (Tb3+) conjugates, respectively. Hg2+ ions induce T33 to form folded structures, leading to increased fluorescence of the T33/TOTO-3 conjugates. Because Pb2+ ions compete with Tb3+ ions to form complexes with G33, the extent of formation of the G33–Tb3+ complexes decreases upon increasing the Pb2+ concentration, leading to decreased fluorescence at 545nm when excited at 290nm. To minimize interference from Hg2+ ions during the detection of Pb2+ ions, we conducted two-step fluorescence measurements; prior to addition of the G33/Tb3+ probe, we recorded the fluorescence of a mixture of the T33/TOTO-3 conjugates and Hg2+ ions. The fluorescence signal obtained was linear with respect to the Hg2+ concentration over the range 25.0–500nM (R2=0.99); for Pb2+ ions, it was linear over the range 3.0–50nM (R2=0.98). The limits of detection (at a signal-to-noise ratio of 3) for Hg2+ and Pb2+ ions were 10.0 and 1.0nM, respectively. Relative to other techniques for the detection of Hg2+ and Pb2+ ions in soil and water samples, our present approach is simpler, faster, and more cost-effective.

An optical-logic system based on cationic conjugated polymer/DNA/intercalating dyes assembly for label-free detection of conformational conversion of DNA i-motif structure

In this paper, we described our strategy for the design, construction, and characterization of a novel supramolecular optical-logic system based on cationic conjugated polymer/DNA/intercalating dyes assembly. Multiple logic gates operating in parallel were simulated by taking advantage of the pH-driven conformational conversion of DNA i-motif structure and the two-step fluorescence resonance energy transfer (FRET) process in this assembly. This logic system does not require any chemical modification or oligonucleotide labeling, which offers the advantages of simplicity and cost efficiency; and it can be switched back and forth by addition of H+ and OH−, which makes the logic gates operate easily and feasible to realize system reset. The strategy also gives rise to a new method for label-free detection of conformational conversion of DNA i-motif structure by working in a simple “mix-and-detect” manner, which provides some inspiration for the investigation of other biomolecular conformational conversions upon environmental changes or binding to their targets.

Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins

There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay option - indirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD ≤ 0.5%, LOQ ≤ 0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.

A Luciferase/Single‐Walled Carbon Nanotube Conjugate for Near‐Infrared Fluorescent Detection of Cellular ATP

Take the tube: The title conjugate provides spatiotemporal information of ATP in living cells by a two-step fluorescence quenching mechanism. OxyLrin, which is generated enzymatically in the reaction between ATP and D-luciferin (Lrin), is adsorbed onto the nanotubes and quenches their fluorescence (see picture). The sensor is highly selective toward ATP.

Role of spacer in single- or two-step FRET: studies in the presence of two connected cryptands with properly chosen fluorophores

Two cryptand molecules are connected via p-xyloyl, benzene-1,4-dicarbolyol and 9,10-dimethylene anthracene. Each cryptand is further derivatized with fluorophores such that electronic absorption of one fluorophore overlaps emission of the other. This way, three different systems L(1), L(2) and L(3) have been synthesized to get a better view of the effect on the distance in single- and two-step fluorescence resonance energy transfer (FRET) process. These molecules are probed for FRET in the presence of a metal ion as input. L(1) and L(2) exhibit poor performance in single step FRET while in the case of L(3), a large two-step FRET process is operational with Cu(II) or Hg(II) as input.

Non-natural amino acid fluorophores for one- and two-step fluorescence resonance energy transfer applications

Fluorescence resonance energy transfer (FRET) provides a powerful means to study protein conformational changes. However, the incorporation of an exogenous FRET pair into a protein could lead to undesirable structural perturbations of the native fold. One of the viable strategies to minimizing such perturbations is to use non-natural amino acid-based FRET pairs. Previously, we showed that p-cyanophenylalanine (Phe CN) and tryptophan (Trp) constitute such a FRET pair, useful for monitoring protein folding–unfolding transitions. Here we further show that 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) can also serve as a FRET acceptor to Phe CN, and the resultant FRET pairs offer certain advantages over Phe CN–Trp. For example, the fluorescence spectrum of 7AW is sufficiently separated from that of Phe CN, making it straightforward to decompose the FRET spectrum into donor and acceptor contributions. Moreover, we show that Phe CN, Trp, and 7AW can be used together to form a multi-FRET system, allowing more structural information to be extracted from a single FRET experiment. The applicability of these FRET systems is demonstrated in a series of studies where they are employed to monitor the urea-induced unfolding transitions of the villin headpiece subdomain (HP35), a designed ββα motif (BBA5), and the human Pin1 WW domain.

Cryptand cage: perfect skeleton for transition metal induced two-step fluorescence resonance energy transfer

Attachment of three different fluorophores to an aza-oxa cryptand (L) gives a transition-metal ion induced two-step fluorescence resonance energy transfer system.

Two-Step Fluorescence Screening of CD21-Binding Peptides with One-Bead One-Compound Library and Investigation of Binding Properties ofN-(2-Hydroxypropyl)methacrylamide Copolymer−Peptide Conjugates

Using the one-bead one-compound (OBOC) combinatorial method, four heptapeptide ligands of CD21 receptor, a cell surface marker of malignant B cell lymphoma, were identified with an innovative two-step fluorescence screening method to overcome the limitation caused by autofluorescence of TentaGel resin. The binding affinities of selected peptides, YILIHRN (B1), PTLDPLP (B2), and LVLLTRE (B3), were in the micromolar region as determined by a fluorescence quenching assay. Peptide B1 was conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via spacers of different lengths, composed of one to four repeats of the 8-amino-3,6-dioxaoctanoic acid (A) group. The evaluation of the biorecognizability of HPMA copolymer-B1 conjugates by the CD21 receptor revealed that increasing the number of repeats of A in the spacer from one to three resulted in continuous improvements in the biorecognition by the CD21 receptor; the increase from three to four repeats showed no significant effect. This work showed the potential of the OBOC combinatorial approach to select peptide ligands as targeting moieties for CD21 specific polymeric drug carriers.

Vibrational Dynamics of Anthracene in Liquid Solution Studied by Picosecond IR/UV Spectroscopy with Polarization Resolution

Two-step fluorescence excitation is carried out with tunable pulses in the infrared in the range 3000−3100 cm-1 and in the near ultraviolet. The anthracene molecules are promoted close to the bottom of the first excited singlet state via excited vibrational levels in the electronic ground state, the symmetry properties and dynamics of which are studied. The polarization dependence of the excitation spectra is studied theoretically and experimentally to determine the relative orientation of the involved transition dipoles. Comparison with conventional absorption spectra yields information on the symmetry and assignment of the vibrational levels, not accessible in previous work without polarization resolution. Evidence is presented for the prominent role of overtone levels, populated by weak IR absorption and also energy redistribution from CH-stretching modes. The polarization dependence is also applied to separate reorientational and vibrational relaxation and to study the vibrational dynamics unambiguous...