Generation Of Fluorescent Signal Research Articles

Highly sensitive fluorescence-linked immunosorbent assay based on aggregation-induced emission luminogens incorporated nanobeads

Aggregation-induced emission luminogens (AIEgens) have attracted considerable interest for application towards the development of various biosensors due to their unique optical properties. However, the major challenge associated with generating a suitable fluorescent signal for constructing an AIEgens-based immunoassay platform, is the complex surface modification and additional chemical reaction required to activate the AIE process. This work reports a novel AIEgens nanobeads-based fluorescence-linked immunosorbent assay (FLISA) platform wherein the fluorescent labels are hexaphenylsilole (HPS) nanobeads, which were synthesized through Shirasu porous glass (SPG) membrane emulsification method and could provide a strong, direct fluorescent signal without any pretreatment. Moreover, the particle-based signal amplification effect affords this platform significantly improved detection sensitivity for carcinoembryonic antigen (CEA) quantitation. Compared to FLISA which uses R-phycoerythrin (PE) or commercial green QDs nanobeads as fluorescent labels, this AIEgens nanobeads-based FLISA platform exhibits detection sensitivity improved up to 45-fold and 12-fold, respectively. Clinical validation experiments applying this AIEgens nanobeads-based FLISA immunoassay platform to analyze human serum samples produce results consistent with those obtained by the clinical gold-standard method, electrochemiluminescence immunoassay (ECLIA). The strong photobleaching resistance and excellent fluorescent stability of the HPS nanobeads negate the need for light shielding, which improves the efficiency and makes the operating conditions more comfortable. Thus, this AIEgens nanobeads-based FLISA platform, with attractive features including direct fluorescent signal generation and significant signal amplification, creates a new, versatile route for the application of AIEgens in biosensors and clinical diagnosis.

A label-free fluorescence method based on terminal deoxynucleotidyl transferase and thioflavin T for detecting prostate-specific antigen.

Prostate-specific antigen (PSA) is the only biomarker for the diagnosis of prostate cancer. So the PSA screening test is very important due to the high occurrence of prostate cancer in men. In this work, a label-free fluorescent method was developed based on terminal deoxynucleotidyl transferase (TdT) and G-quadruplex-thioflavin T complex for detecting PSA. In the absence of PSA, the PSA aptamer can be used as the primer for TdT extension reactions, resulting in the formation of G-quadruplexes and generation of strong fluorescent signals. After the addition of PSA, the PSA-aptamer complex prevented the TdT extension reaction due to steric hindrance, thus resulting in a poor fluorescent signal. The assay showed a wide linear range (0.1 to 80pg/mL) and a detection limit of 0.086pg/mL (S/N = 3). It also has good specificity for PSA determination and gives satisfactory results when applied to biological samples. Conceivably, its merits such as good selectivity and high sensitivity indicate that the proposed method has a promising application potential in the clinical diagnosis and treatment of prostate cancer. Graphical abstract.

Visual Electrofluorochromic Detection of Cancer Cell Surface Glycoprotein on a Closed Bipolar Electrode Chip.

This work reports an electrofluorochromic strategy on the basis of electric field control of fluorescent signal generation on bipolar electrodes (BPEs) for visualizing cancer cell surface glycoprotein (mucin 1). The device included two separate cells: anodic sensing cell and cathodic reporting cell, which were connected by a screen-printing electrode patterned on poly(ethylene terephthalate) (PET) membrane. In the sensing cell, anti-MUC1 antibody immobilized on a chitosan-multiwalled carbon nanotube (CS-MWCNT)-modified anodic BPE channel was used for capturing mucin-1 (MUC1) or MCF-7 cancer cells. Then ferrocene (Fc)-labeled mucin 1 aptamers were introduced through hybridization. Under an applied voltage, the ferrocene was oxidized and the electroactive molecules of 1,4-benzoquinone (BQ) in the cathodic reporting cell were reduced according to electroneutrality. This produced a strongly basic 1,4-benzoquinone anion radical (BQ•-), which turned on the fluorescence of pH-responsive fluorescent molecules of (2-(2-(4-hydroxystyryl)-6-methyl-4 H-pyran-4-ylidene)malononitrile) (SPM) coexisting in the cathode reporting cell for both spectrophotometric detection and imaging. This strategy allowed sensitive detection of MUC1 at a concentration down to 10 fM and was capable of detecting a minimum of three MCF-7 cells. Furthermore, the amount of MUC1 on MCF-7 cells was calculated to be 6.02 × 104 molecules/cell. Our strategy also had the advantages of high temporal and spatial resolution, short response time, and high luminous contrast and is of great significance for human health and the promotion of life science development.

A pH-Activatable nanoparticle for dual-stage precisely mitochondria-targeted photodynamic anticancer therapy

Mitochondria-targeted photodynamic therapy (PDT) has emerged as one of the most efficient antitumor strategies. However, the therapeutic outcome of mitochondria-targeted PDT nanocarriers has been hampered by its poor capability of endosome escape and always-ON mode which induces normal tissue damage. To tackle these limitations, herein a novel pH-activatable nanoparticle is developed with dual-stage targeting capacity of early endosome and mitochondria for exponential activation of fluorescent signals and photodynamic efficacy. This nanoparticle is composed of pH-responsive mPEG-b-PDPA-Cy7.5 fluorescent copolymer and mitochondria-targeted photosensitizer (TPPa). The TPPa-encapsulated nanoparticles (M-TPPa) exhibit 111- and 151-fold enhancement in fluorescent signal and singlet oxygen generation (SOG) on encounting acidic pH environment, respectively. The M-TPPa can be quickly endocytosed by cancer cells and immediately dissociate at acidic early endosome to activate fluorescent signals and photoactivity. Subsequently, the activated TPPa quickly translocates from early endosome to mitochondria. Under laser irradiation, singlet oxygen could be generated in mitochondria, inducing intrinsic apoptosis in human HO8910 ovarian cancer cells. M-TPPa also exhibits high tumor imaging contrast and remarkable inhibition on tumor progression without obvious toxicity in HO8910-tumor bearing mice. Therefore, the rationally designed nanoparticles, with precise dual-targeting of distinct organelles and theranostic signal amplification, provides a promising strategy for efficient cancer treatment.

Proximity hybridization-induced on particle DNA walker for ultrasensitive protein detection

A simple proximity hybridization-induced on particle DNA walker was designed for ultrasensitive detection of proteins, for example platelet-derived growth factor (PDGF-BB) secreted by cancer cells, in which the DNA walker was activated by specific target binding and powered by an enzymatic cleavage to produce amplified signal. High-density FAM-labeled hairpin oligonucleotides (FAM-DNA1) were functionalized on AuNPs to construct three-dimensional (3D) DNA tracks. The specific binding of PDGF-BB with two aptamer probes (DNA3 and DNA4) led to the proximity hybridization-induced DNA displacement and the free of DNA walker (DNA2) to perform movement on the 3D tracks by an enzymatic cleavage, resulting in the release of massive FAM-DNA1 fragments from the AuNPs and the generation of fluorescent signal. This DNA walker based sensing strategy could detect PDGF-BB in a concentration range of 4 orders of magnitude with a detection limit down to sub-pM level. The practical applicability of the assay was demonstrated by detecting PDGF-BB secreted from MCF-7 cells with satisfactory results. The proposed DNA walker based assay could conveniently detect PDGF-BB with high sensitivity and good accuracy, along with the good extensibility of the assay, showing promise for practical diagnosis.

An Aggregation-Induced Emission-Based Indirect Competitive Immunoassay for Fluorescence "Turn-On" Detection of Drug Residues in Foodstuffs.

A new fluorescent “turn-on” probe-based immunosensor for detecting drug residues in foodstuffs was established by combining the mechanism of aggregation-induced emission (AIE) and an indirect competitive enzyme-linked immunosorbent assay (ELISA). In this study, a luminogen, with negligible fluorescence emission (TPE-HPro), aggregated in the presence of H2O2, and exhibited astrong yellow emission based on its AIE characteristics. This AIE process was further configured into an immunoassay for analyzing drug residues in foodstuffs. In this approach, glucose oxidase (GOx) was used as an enzyme label for the immunoassay and triggered GOx/glucose-mediated H2O2 generation, which caused oxidation of TPE-HPro and a “turn-on” fluorescence response at 540 nm. To quantitatively analyze the drug residues in foodstuffs, we used amantadine (AMD) as an assay model. By combining the AIE-active “turn-on” fluorescent signal generation mechanism with conventional ELISAs, quantifying AMD concentrations in chicken muscle samples was realized with an IC50 (50% inhibitory concentration) value of 0.38 ng/mL in buffer and a limited detection of 0.06 μg/kg in chicken samples. Overall, the conceptual integration of AIE with ELISA represents a potent and sensitive strategy that broadens the applicability of the AIE-based fluorometric assays.

Analyte Regeneration Fluorescent Probes for Formaldehyde Enabled by Regiospecific Formaldehyde-Induced Intramolecularity.

An important challenge for reaction-based fluorescent probes is that they generally require analyte consumption for fluorescence signal generation, thus creating potential perturbation of native analyte homeostasis or change of local concentrations. Herein, we reported two formaldehyde (FA) regeneration fluorescent probes, NAP-FAP-1 and NAP-FAP-2. An unprecedented regiospecific FA-induced intramolecularity strategy is implemented in the probe design, which adopts 3-(benzylamino)-succinimide as the FA-selective reaction group. The probes are able to capture the analyte molecule, induce regiospecific imide bond cleavage, and then release the captured FA molecule with simultaneous fluorescence turn-on response via a unique dual PeT/ICT quenching mechanism. The probes have shown potentials in detection, comparison, and imaging of FA levels intracellularly and inside lysosomes. These features make them useful for the study of FA homeostasis and functions in biological systems with minimal perturbation.

A lateral flow channel immunoassay combining a particle binding zone geometry with nanoparticle labelling amplification

A lateral flow channel immunoassay is described which combines two strategies for signal enhancement. A packed silicagel microparticle matrix, covalently labelled with capture antibodies, was incorporated into a microchannel, providing a detection surface to sample volume ratio at least 20 times the standard wall modified microfluidic channel format. Strong signal amplification was achieved with non-direct fluorescence labelling. Fluorescein diacetate nanoparticles, synthesized by a simple top-down method, increase the number of fluorophores per antibody binding event, compared with a classical molecular fluorescein label. The fluorescent signal generation mechanism, triggered by the addition of a solution of NaOH:DMSO, was optimised to produce highly fluorescent uranin. The packed channel immunoassay showed a linear relationship between fluorescence intensity and antibody concentration for detection of 0.4–1.67 nM in a model system of rabbit polyclonal antibodies (RbIgG) and goat anti-rabbit polyclonal antibodies (Gt-α RbIgG), potentially enabling this format to achieve semi-quantitative or quantitative measurement of multiple target analytes, where packed silica zones modified with different capture antibodies are incorporated into the same microchannel system.

Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides

In this study we optimised the fluorescence signal generation of contact quenched universal reporter oligonucleotides.

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

Inkjet-printed barcodes for a rapid and multiplexed paper-based assay compatible with mobile devices.

This study reports a simple, rapid, low-cost, robust, and multiplexed barcoded paper-based assay (BPA) compatible with mobile devices. An inkjet printer and an XYZ dispensing platform were used to realize mass-manufacturing of barcoded paper-based analytical devices (BPADs) with high precision and efficiency. We designed a new group of barcodes and developed an application (APP) for the reading of the new code. The new barcodes possess a 16 times higher coding capacity than the standard Codabar code in our experiment on drug residue detection. The BPA system allows applications in the assays of blood-transmitted infections, drug residues in milk and multiplex nucleic acids. The whole detection process and the readout of the results can be completed within 10 minutes. The limit of detection for enrofloxacin (ENR) (8 ng mL-1) satisfies the requirements of drug residue monitoring. Its high rapidity, simplicity, efficiency and selectivity make the BPA system extremely suitable to be applied in rapid and on-site detection.

Use of models of secondary X-ray fluorescence spectra to determine the measurement conditions in X-ray spectral methods of material analysis

The principles of construction and the components of a model of X-ray fluorescence signal generation are considered in this paper. It is shown that the developed model describes the X-ray spectra of various materials rather well. Issues with choosing the optimal parameters of measurements in the quantitative X-ray fluorescence analysis of individual samples using the constructed models are discussed.

Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy

Deep-tissue multiphoton microscopy (MPM) enables noninvasive optical imaging into the deep regions of the tissue in animal models in vivo . High-order MPM techniques, such as three- and four-photon fluorescence microscopy at the 1700-nm window, are emerging as promising imaging techniques for deeper penetration. Currently, signal depletion at large imaging depth sets the depth limit for these imaging techniques. As a result, how to further boost signal level is the key to achieving a larger imaging depth. Contrary to the previous thought that overfilling the back aperture of the objective yields the highest multiphoton fluorescence signal, in this paper, through numerical simulation, we show that, due to the effect of exponential decay of the excitation beam, the signal generation is maximized for certain underfilling of the back aperture of the objective lens. This will provide a simple strategy for signal generation maximization in deep-tissue MPM and potentially enables deeper imaging penetration into the tissue.

In situ formation of fluorescent copper nanoparticles for ultrafast zero-background Cu2+ detection and its toxicides screening

Copper pollution has become more and more serious in modern society as the increasing industrial emission and the acid mine drainage, and exposure to excess copper can result in damage to living organisms. Thus, the development of efficient strategy for copper ion (Cu2+) detection is very essential and significant. Here, a high-efficiency fluorescent method is proposed for Cu2+ monitoring. The detection mechanism is based on the in situ formation of fluorescent copper nanoparticles (CuNPs). When the water sample is polluted by Cu2+, fluorescence emission of CuNPs can be observed by a one-step manner, and the emission intensity is proportional to Cu2+ concentration. Attractively, besides its advantages in operation and good detection capability, the generation of fluorescent signal is ultrafast, with a good signal response in 1min; and there is no interference from background and other ions due to the in situ formation of signal unit. By virtue of its advantages, this strategy has been used to detect Cu2+ from polluted tap and river water samples, good performances demonstrate that the proposed method can be practically applied for Cu2+ monitoring in real drinking and environmental water. Simultaneously, great potential for Cu2+ toxicides screening has been verified by direct analysis of the effects of different model molecules on Cu2+, which will contribute to Cu2+-related sewage treatment and medical therapy.

Designing Theranostic Agents Based on Pluronic Stabilized Gold Nanoaggregates Loaded with Methylene Blue for Multimodal Cell Imaging and Enhanced Photodynamic Therapy.

At present, multifunctional noble metal-based nanocomposites are extensively investigated for their potential in performing cellular imaging, diagnostics, and therapy by integration of unique plasmonic properties with the spectroscopic expression and therapeutic activity of appropriate drug. In this work, we report the fabrication of 3-dimensional (3-D) close-packed nanoassemblies of gold nanoparticles by controlling the aggregation of individual nanoparticles in solution and subsequent stabilization of formed aggregates by Pluronic block copolymer (F127) coating. Besides conferring high stability, Pluronic mediates the loading of Methylene Blue (MB) molecules which exhibit interesting spectroscopic and photochemical properties to be employed as both optical label and photosensitizing drug. Indeed, here we demonstrate the pertinence of the fabricated nanoassemblies to provide optical imaging of murine colon carcinoma cells (C-26) via both Raman and fluorescence signals collected from MB molecules, specifically by using scanning confocal surface-enhanced resonant raman spectroscopy (SERRS) and fluorescence lifetime imaging microscopy (FLIM) techniques. The specific configuration of as fabricated nanoassemblies allows a small population of MB molecules to be located in very small areas between the aggregated nanoparticles ("hot spots") to provide SERRS signal while the other population remains captured in Pluronic coating and preserves both its fluorescence signal and singlet-oxygen generation capability. Remarkably, we demonstrate an enhanced photodynamic therapeutic activity of MB-loaded gold nanoaggregates against murine colon carcinoma cells (C-26), as compared to the free photosensitizer. To our knowledge, this is the first report on plasmonic nanoplatforms conveying photosensitizing drug into cells to operate as optical label via both SER(R)S and FLIM and to perform enhanced photodynamic therapy.

A hybrid chimeric system for versatile and ultra-sensitive RNase detection.

We developed a new versatile strategy that allows the detection of several classes of RNases (i.e., targeting ss- or ds-RNA, DNA/RNA hetero-hybrid or junctions) with higher sensitivity than existing assays. Our two-step approach consists of a DNA-RNA-DNA chimeric Hairpin Probe (cHP) conjugated to magnetic microparticles and containing a DNAzyme sequence in its terminal region, and molecular beacons for fluorescence signal generation. In the first step, the digestion of the RNA portion of the cHP sequences in presence of RNases leads to the release of multiple copies of the DNAzyme in solution. Then, after magnetic washing, each DNAzyme molecule elicits the catalytic cleavage of numerous molecular beacons, providing a strong amplification of the overall sensitivity of the assay. We successfully applied our approach to detect very low concentrations of RNase A, E. coli RNase I, and RNase H. Furthermore, we analyzed the effect of two antibiotics (penicillin and streptomycin) on RNase H activity, demonstrating the applicability of our strategy for the screening of inhibitors. Finally, we exploited our system to detect RNase activity directly in crude biological samples (i.e., blood and saliva) and in cell culture medium, highlighting its suitability as cheap and sensitive tool for the detection of RNase levels.

Fluorescent microRNA biosensors: a comparison of signal generation to quenching

Many microRNA biosensor platforms regard fluorescence signal generation as superior to quenching; however, the two methods are comparable rather than competitive.

Mechanism for laser-induced fluorescence signal generation in a nanoparticle-seeded flow for planar flame thermometry

The mechanism of atomic indium generation for laser-induced fluorescence (LIF) of indium from laser ablation seeding was investigated in a hydrogen/nitrogen non-premixed flame. The morphology and particle size distributions of the ablation products were examined with scanning electron microscopy and transmission electron microscopy. These investigations show that the ablation products comprise complex agglomerates of nano-sized primary particles of indium compounds and micron-sized spherical indium beads. Images of the atomic indium LIF, Mie scattering of ablation products and natural fluorescence emission of indium in the flame were recorded to investigate the mechanism of fluorescence signal generation. The relative contribution of natural fluorescence emission of indium towards the total indium fluorescence signal was assessed by comparing these images. These images also reveal the evolution of ablation products through the flame structure and the correlation between LIF signal and ablation products. It is found that the LIF signal generation is associated with the vapourisation of indium nanoparticles into the gas phase by thermal decomposition in the flame. A further mechanism for thermal decomposition of the nanoparticles was also identified, that of heating the ablation products by in situ laser ablation. This was assessed by means of a second laser, introduced prior to the excitation laser, to reveal that the LIF signal can be enhanced by in situ laser ablation, particularly in the upstream regions of the flame. These findings supersede the mechanism deduced previously by the authors that neutral atomic indium can survive a convection time of the order of tens of seconds and be directly seeded into reacting or non-reacting flows. The possible influences of laser ablation seeding on the nonlinear two-line atomic fluorescence thermometry technique were also assessed.

A general strategy to create RNA aptamer sensors using "regulated" graphene oxide adsorption.

Aptamers have been used as molecular recognition elements for sensor development in combination with graphene oxide (GO), a nanomaterial with properties including fluorescence quenching, and selective adsorption of single-stranded nucleic acids. However, previous sensor designs based on aptamer-GO adsorption have not demonstrated wide applicability, and few studies have explored the potential of RNA aptamers. Herein, we present a new sensing strategy based on "regulated" GO adsorption that can accommodate various RNA aptamers. First, adsorption of a fluorophore-labeled RNA aptamer to GO results in fluorescence quenching due to close proximity of the fluorophore to GO. The addition of a complementary, "blocking" DNA strand (BDNA) that hybridizes to the 3'-end of the aptamer, weakens aptamer-GO interaction, and enables increased fluorescent signal generation upon the addition of target, as the sensing system becomes completely separated from GO. Our findings can be applied toward different aptamers, and adapted to enhance generality of existing sensing applications.

Intra- and Interlayer Energy Transfer in Planar Systems Based on Amphiphilic Naphthalimide Derivatives

The present work considers the possibility of increasing the information signal of thin-film sensitive element of the sensor based on amphiphilic crown substituted derivative of 4-(acylamino)-1,8-naphthalimide via employment of resonance photoexcitation energy transfer process. In such system, information signal is the fluorescence of acceptor fluorophore (4-alkylamino-1,8-naphthalimide), which is excited upon non-radiative transfer of the energy from excited donor fluorophore (crown containing derivative of 4-(acylamino)-1,8-naphthalimide), crown ether fragment of which is “occupied” by coordination interaction with specific cation and thus does not participate in rapid relaxation of the excitation by the photoinduced electron transfer mechanism. For realization of this goal, the synthesis of novel amphiphilic naphthalimide derivatives is carried out; conditions for obtaining of stable monolayers and Langmuir-Blodgett films based on them are studied. It is established that azadithiacrown substituted amphiphilic fluoroionophore in monolayers at air/mercury and silver perchlorate aqueous solutions interface retains the ability for efficient binding of Ag + and Hg 2+ known for its solutions. Described principle of generation of fluorescent signal using mixed monolayer containing the donor-acceptor couple allows one to increase the value of optical response of the monolayer to the presence of assessed cations by 2.5 times. However, the monolayer fluorescence intensity remains too low for practical application. In order to optimize the conditions of the energy transfer process in more practically important solid-state sensory elements we studied the mechanism of non-radiative photoexcitation energy transfer in the multilayer thin-film system based on amphiphilic naphthalimide derivatives obtained by Langmuir-Blodgett (LB) technique. It is shown that this problem can be solved by regulation of the distance between donor and acceptor layers of LBF. Optimal thickness of the separating layer of stearic acid, which for this system amounted to 7 nm, was determined experimentally. Obtained results open up new perspectives for increase of the information signal reading efficiency, which should find application in development of sensitive elements of sensor devices.