A validated quantitative method for the assessment of neuroprotective barrier impairment in neurodegenerative disease models.

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are highly specialized structures that limit molecule entry from the blood and maintain homeostasis within the central nervous system (CNS). BBB and BSCB breakdown are associated with multiple neurodegenerative diseases. Given the key role of neuroprotective barrier impairment in neurodegeneration, it is important to identify an effective quantitative method to assess barrier integrity in animal models. In this study, we developed and validated a quantitative method for assessing BBB and BSCB integrity using sodium fluorescein, a compound that outperformed other fluorescent dyes. We demonstrated using this method that multiple CNS regions progressively increase in permeability in models of Huntington's disease and amyotrophic lateral sclerosis, whereas biphasic disruption occurred in a mouse model of Alzheimer's disease with disease progression. Collectively, we report a quantitative fluorometric marker with validated reproducible experimental methods that allows the effective assessment of BBB and BSCB integrity in animal models. This method could be useful to further the understanding of the contribution of these neuroprotective barriers to neurodegeneration processes.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL-1 with a large dynamic range from 10 pg mL-1 to 1 µg mL-1.

Determination of iron(II) and iron(III) via static quenching of the fluorescence of tryptophan-protected copper nanoclusters.

"Tryptophan-coated blue fluorescent copper nanocluster (CuNC@Trp) was prepared by a strategy where Trp acts as both the reducing and capping agent. The fluorescence of the CuNC, with excitation/emission peaks at 340/405 nm, is selectively quenched by iron(II) and iron(III) ions. Studying the mechanism of this interaction revealed that Fe2+ and Fe3+ ions can make a ground state complex with the protecting ligand which can result in quenching of the cluster emission. Structural and optical properties of the modified CuNC were investigated by ESI-MS, DLS, TEM, UV-vis and photoluminescence. The effects of pH value and temperature, time of interaction, and cluster volume were optimized. Under optimized conditions, the probe response is linear in concentration range of 10-1000 µM for Fe(II) and Fe(III) with the relative standard deviations of 0.13 and 0.14% (n = 5) respectively. The respective limits of detection are 3.0 and 2.2 µM. The method was successfully used for determination of trace amount of both ions in spiked water, blood and iron supplement tablets. The results were in good agreement with those obtained by the ICP-AES method." Graphical abstractThe scheme represents the synthesis of CuNC@Trp at basic conditions and at elevated temperature. The emission of the cluster decreases due to static quenching of fluorescence by iron ions.

A terbium(III)-functionalized zinc(II)-organic framework for fluorometric determination of phosphate.

A terbium(III)-functionalized zinc(II)-organic framework (Tb-MOF-Zn) is shown to be a viable fluorescent probe for phosphate. The organic ligands 4,4',4″-[((2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene))tris(oxy)]tribenzoic acid (H3L3) contains multiple carboxyl groups that can react with zinc(II) to yield tubular MOF-Zn. The MOF-Zn was further functionalized with Tb(III) to produce a lanthanide composite of type Tb-MOF-Zn which displays strong fluorescence with excitation/emission maxima at 285/544 nm. Fluorescence is quenched by phosphate because of the specific interaction with Tb(III) in Tb-MOF-Zn. The concentration of Tb-MOF-Zn, reaction time and pH value of the solution were optimized. Fluorescence drops linearly in the 0.01 to 200.0 µM phosphate concentration range, and the detection limit is 4.0 nM. The fluorescent probe was also used to prepare a microdot array on a glass slide for visual detection of phosphate under illumination with UV light. Graphical abstractA terbium(III) functionalized zinc(II)-organic framework was synthesized and used as fluorescent probe for determination of phosphate ions.

Hemin@carbon dot hybrid nanozymes with peroxidase mimicking properties for dual (colorimetric and fluorometric) sensing of hydrogen peroxide, glucose and xanthine.

The multifunctional hemin@carbon dot hybrid nanozymes (hemin@CD) with simultaneous peroxidase-like activity and fluorescence signalling property was prepared for the first time. Based on these properties, hemin@CD was applied to develop a dual-channel fluorescent probe for H2O2 and H2O2-based biocatalytic systems. By virtue of the peroxidase-like activity, hemin@CD can catalyze the oxidative coupling of 4-aminoantipyrine with phenol in the presence of H2O2 to form a pink-red quinoneimine dye with a maximum absorbance at 505 nm. Under the excitation wavelength of 480 nm, the green fluorescence of hemin@CD peaks at 540 nm and is quenched by the generated quinoneimine dye due to an inner filter effect, and also by H2O2 because of dynamic quenching. Thus, a colorimetric and fluorimetric dual-channel optical probe for H2O2 is obtained. Due to the glucose/xanthine transformations under formation of H2O2 by the relevant oxidase catalysis, the probe can be applied for detection of glucose and xanthine. The colorimetric detection limits for H2O2, glucose and xanthine are 0.11, 0.15, 0.11 µM, and the and fluorimetric detection limits are 0.15, 0.15, 0.12 µM, respectively. Graphical abstractSchematic representation of the colorimetric and fluorimetric dual probe for H2O2, glucose and xanthine based on the multifunctional emin@carbon dot) hybrid nanozymes with simultaneous peroxidase-like activity and fluorescence signalling property.

Fluorometric determination of lead(II) by using aptamer-functionalized upconversion nanoparticles and magnetite-modified gold nanoparticles.

A fluorescent nanoprobe for Pb(II) has been developed by employing aptamer-functionalized upconversion nanoparticles (UCNPs) and magnetic Fe3O4-modified (MNPs) gold nanoparticles (GNPs). First, aptamer-functionalized UCNPs and aptamer-functionalized magnetic GNPs were synthesized to obtained the fluorescent nanoprobe. The particles were combined by adding a complementary ssDNA. In the absence of Pb(II), the UCNPs, MNPs and GNPs are linked via complementary base pairing. This led to a decrease in the green upconversion fluorescence peaking at 547 nm (under 980 nm excitation). In the presence of Pb(II), the dsDNA between UCNPs and MNPs-GNPs is cleaved, and fluorescence recovers. This effect allows Pb(II) to be quantified, with a wide working range of 25-1400 nM and a lower detection limit of 5.7 nM. The nanoprobe gave satisfactory results when analyzing Pb(II) in tea and waste water. Graphical abstractSchematic representation of fluorescent nanoprobe based on fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (GNPs)-Fe3O4 magnetic nanoparticles (MNPs) for detection of Pb2+.

Fluorimetric determination of histidine by exploiting its inhibitory effect on the oxidation of thiamine by cobalt-containing Prussian Blue nanocubes.

A fluorometric assay for histidine (His) is described. It is based on the inhibitory effect of His on nanocubes consisting of cobalt-containing Prussian Blue analog (CoFe NCbs), which have a strong oxidation effect on thiamine (THI) in the presence of NaOH. THI is nonfluorescent but the oxidized form (thiochrome; ThC) has a strong blue fluorescence, with excitation/emission maxima at 370/445 nm. His inhibits the oxidation effect of the CoFe NCbs due to the strong interaction between its imidazole side chain and the amino groups of the CoFe NCbs. This method is fast and has good sensitivity and selectivity. The lower detection limit is 14.3 nM of His, the linear range extends from 0.05 to 2.5 µM, and the relative standard deviation is calculated to be 1.5%. The method was successfully employed to quantify His in spiked serum samples. Graphical abstractSchematic representation of cobalt-containing Prussian Blue nanocubes (CoFe NCbs)-thiamine (THI)-based fluorometric assay for Histine (His). His inhibits the generation of thiochrome (ThC; the oxidized form of THI). The detection limit is 14.3 nM with the linear range of 0.05-2.5 µM.

A "turnon" aptasensor for simultaneous and time-resolved fluorometric determination of zearalenone, trichothecenes A and aflatoxin B1 using WS2 as a quencher.

A "turn on" time-resolved fluorometric aptasensor is described for the simultaneous detection of zearalenone (ZEN), trichothecenes A (T-2), and aflatoxin B1 (AFB1). Multicolor-emissive nanoparticles doped with lanthanide ions (Dy3+, Tb3+, Eu3+) were functionalized with respective aptamers and applied as a bioprobe, and tungsten disulfide (WS2) nanosheets are used as a quencher of time-resolved fluorescence. The assay exploits the quenching efficiency of WS2 and the interactions between WS2 and the respective DNA aptamers. The simultaneous recognition of the three mycotoxins can be performed in a single solution. In the absence of targets, WS2 is easily adsorbed by the mixed bioprobes via van der Waals forces between nucleobases and the WS2 basal plane. This brings the bioprobe and WS2 into close proximity and results in quenched fluorescence. In the presence of targets, the fluorescence of the bioprobes is restored because the analytes react with DNA probe and modify their molecular conformation to weaken the interaction between the DNAs and WS2. Under the optimum conditions and at an excitation wavelength of 273 nm, the time-resolved fluorescence intensities (peaking at 488, 544 and 618 nm and corresponding to emissions of Dy3+, Tb3+ and Eu3+) were used to quantify ZEN, T-2 and AFB1, respectively, with detection limits of 0.51, 0.33 and 0.40 pg mL-1 and a linear range from 0.001 to 100 ng mL-1. The three mycotoxins can be detected simultaneously without mutual interference. The assay was applied to the quantification of ZEN, T-2 and AFB1 in (spiked) maize samples. This homogeneous aptamer based assay can be performed within 1 h. Conceivably, it can become an alternative to other heterogeneous methods such as the respective enzyme-linked immunosorbent assays. Graphical abstract Schematic presentation of an aptasensor for simultaneous detection of zearalenone, trichothecenes A and aflatoxin B1 using aptamer modified time-resolved fluorescence nanoparticles as signalling probes and tungsten disulfide as the quencher. This assay shows lower detection limit and requires no washing steps.

Fluorometric determination of microRNA by using an entropy-driven three-dimensional DNA walking machine based on a catalytic hairpin assembly reaction on polystyrene microspheres.

An entropy-driven 3-D DNA walking machine is presented which involves catalytic hairpin assembly (CHA) for detection of microRNA. A 3-D DNA walking machine was designed that uses streptavidin-coated polystyrene microspheres as track carriers to obtain reproducibility. The method was applied to microRNA 21 as a model analyte. Continuous walking on the DNA tracks is achieved via entropy increase. This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cycling of microRNA 21. The release of massive auxiliary strands from ternary DNA substrates induces the CHA. This is accompanied by in increase in fluorescence, best measured at excitation/emission wavelengths of 480/520 nm. On account of entropy-driven reaction, the assay is remarkably selective. It can differentiate microRNA 21 from homologous microRNAs in giving a signal that is less than 5% of the signal for microRNA 21 except for microRNA-200b. The assay works in the 50 pM to 20 nM concentration range and has a 41 pM detection limit. The method displays good reproducibility (between 1.1 and 4.2%) and recovery (from 99.8 to 104.0%). Graphical abstract An entropy-driven 3-D DNA walking machine is described. It is based on the use of polystyrene microspheres and of a catalytic hairpin assembly reaction for sensitive microRNA detection. Figure Notes: AS represents auxiliary strand; S represents substrate strand; LS represents link strand; F represents fuel nucleic acid; RepF represents nucleic acid labeled with FAM; RepQ represents nucleic acid labeled with BHQ1.

Boron doped carbon dots as a multifunctional fluorescent probe for sorbate and vitamin B12.

Boron doped carbon dots (B-CD) were synthesized by a one-step hydrothermal method using phenylboronic acid as the starting material. They have an average size of about 3.3 nm, with excitation/emission wavelength of 247/323 nm and a quantum yield of 12%. The B-CD is shown to be viable fluorescent probe for sorbate (PS) and vitamin B12 (VB12). The fluorescence (FL) of the B-CD is quenched in the presence of PS or VB12 mainly coming from inner filter effect (IFE), but Förster resonance energy transfer (FRET) from the B-CD (as a donor) to PS/VB12 (as an acceptor) cannot be excluded. The probe enables PS to be detected by fluorometry with a linear response in the 0.20-24 µM concentration range and a 6.1 nM detection limit (at 3σ/slope). For VB12, the data are 0.20-30 µM and 8.0 nM. Graphical abstract Boron doped carbon dots (B-CD) as a probe was prepared by phenylboronic acid as single starting material via one-step hydrothermal method, which has remarkable selectivity and high sensitivity for monitoring PS/VB12. The fluorescence quenching of B-CD by PS/VB12 mainly comes from inner filter effect.

Fluorometric determination of glucose based on a redox reaction between glucose and aminopropyltriethoxysilane and in-situ formation of blue-green emitting silicon nanodots.

A method is described for fluorometric detection of glucose. It is based on the finding that silicon nanodots (SNDs) are formed from glucose and aminopropyltriethoxysilane (APTES) under mild experimental conditions. The SNDs thus formed have an average diameter of ∼2 nm, exhibit good water dispersibility, blue fluorescence (with excitation/emission maxima at 410/475 nm), broad pH tolerance, and are photostable. The assay was applied to the quantification of glucose with high sensitivity, good specificity, and over a wide detection range (from 10 µM to 0.9 mM). It was applied to the determination of glucose in spiked serum samples and gave satisfactory results and recoveries. Graphical abstract Schematic presentation of serum glucose detection based on a redox reaction between glucose and aminopropyltriethoxysilane and in-situ formation of blue-green emitting silicon nanodots.

Gold-decorated magnetic nanoparticles modified with hairpin-shaped DNA for fluorometric discrimination of single-base mismatch DNA.

The authors describe the use of gold-decorated magnetic nanoparticles (Au/MNPs) in discriminating DNA sequences with a single-base (guanine) mismatch. The Au/MNPs were characterized through dynamic light scattering, X-ray diffraction, superconducting quantum interference device, and UV/visible spectroscopy. They were then conjugated to a probe oligomer consisting of a hairpin-shaped DNA sequence carrying two signalling fluorophores: fluorescein at its 3' end and pyrene in the loop region. When interacting with the target DNA sequences, the hybridized probe-target duplex renders the pyrene signal (at excitation/emission wavelengths of 345/375 nm) either quenched or unquenched. Quenching (or nonquenching) of the pyrene fluorescence depends on the presence of a guanine (or a nonguanine) nucleotide at the designated polymorphic site. The linear range of hybridization in these Au/MNPs is from 0.1 nM to 1.0 µM of ssDNA. Conceivably, this system may serve as a single-nucleotide polymorphism probe. Graphical Abstract Schematic presentation of probe-conjugated Au/MNP preparation (upper panel) and working principle to discriminate DNA with or without single-base (guanine) mismatch sequences at the polymorphic sites (lower panel). Py denotes pyrene-hooked pyrrolocytidine; F denotes fluorescein.

Sensitive fluorometric determination of glutathione using fluorescent polymer dots and the dopamine-melanin nanosystem.

A bioinspired fluorometric method has been developed for the detection of glutathione (GSH) in biological fluids. It is based on the use of near-infrared fluorescent semiconducting polymer dots (P-dots) and of the dopamine (DA)-melanin nanosystem. The P-dots were prepared from poly(styrene-co-maleic anhydride), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] and the fluorescent dye tetraphenylporphyrin. They have excitation/emission maxima at 458/656 nm, and this enables measurement to be performed with low autofluorescence and scattering background. DA can self-polymerize on the surface of the P-dots to yield a poly-DA coating. This coating, at weak alkaline pH values, causes the quenching of the fluorescence of the P-dots. However, the polymerization of DA is inhibited by GSH. Hence, quenching of fluorescence is prevented. This effect was used to design a fluorometric assay for GSH that has good selectivity and sensitivity. Under optimal conditions, the method has a linear response in the 0.2 to 20 µM GSH concentration range and a 60 nM detection limit. It was successfully applied to the determination of GSH in HepG2 cells and in spiked human serum. Graphical abstract Schematic representation of using a NIR fluorescent P-dots and dopamine (DA)-melanin nanohybrid as a probe for glutathione (GSH) detection. The P-dots were prepared from poly(styrene-co-maleic anhydride) (PSMA), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] (PEPV) and the fluorescent dye tetraphenylporphyrin (TPP).The GSH can inhibit the dopamine self-polymerization and prevented the formation of PDA and fluorescence quenching of P-dots.

Electrostatically controlled fluorometric assay for differently charged biotargets based on the use of silver/copper bimetallic nanoclusters modified with polyethyleneimine and graphene oxide.

An electrostatically controlled fluorometric assay is described that is based on the use of silver/copper bimetallic nanoclusters. The nanoclusters were coated with polyethyleneimine (PEI-Ag/CuNCs). At pH 7.4, these particles are positively charged. Their blue fluorescence (with excitation/emission peaks at 341/464 nm) depends on local pH values and temperature. If graphene oxide (which is negatively charged at pH 7.4) is introduced, the fluorescence of the PEI-Ag/CuNCs is quenched. Based on various electrostatic interactions, three kinds of biomacromolecules were detected by fluorometry. These include (negatively charged) heparin, (positively charged) protamine, and (virtually uncharged) trypsin. Heparin is detected by using GO/PEI-Ag/CuNCs, protamine by using GO/heparin/PEI-Ag/CuNCs, and trypsin by using GO/protamine/heparin/PEI-Ag/CuNC. The detection limits and linear ranges are 4.8 nM and 10-450 nM for heparin, 0.09 µg·mL-1 and 0.25-5 µg·mL-1 for protamine, and 0.03 µg·mL-1 and 0.05-1 µg·mL-1 for trypsin. Zeta potentials of the various substances in the system were determined to elucidate the detection mechanism. Comceivably, the method provides a widely applicable approach for electrostatically controlled biomolecular assays. Graphical abstract Schematic presentation of electrostatically controlled fluorometric assay for the detection of heparin, protamine, and trypsin based on the silver/copper bimetallic nanoclusters modified with polyethyleneimine and graphene oxide.

A turn-on fluorescent probe for vitamin C based on the use of a silicon/CoOOH nanoparticle system.

The authors describe a fluorometric method for the turn-on determination of vitamin C (ascorbic acid). The blue fluorescence of silicon nanoparticles (SiNPs; with excitation/emission maxima at 350/450 nm) is found to be quenched by CoOOH nanoparticles (NPs). In the presence of vitamin C, the CoOOH NPs are decomposed by a redox reaction between the diol group of vitamin C and CoOOH NPs. As a result, fluorescence recovers. On the basis of this finding, a fluorometric method was designed for the turn-on detection of vitamin C. Under optimal conditions, the method has a low detection limit (0.47 µM) and a linear response in the 0.5 µM to 20 µM a concentration range. It was successfully applied to the determination of vitamin C in spiked red grape and orange juice, and in vitamin C tablets. Graphical abstract A target-triggered dissociation of quencher-based strategy for the fluorescence "turn-on" detection of vitamin C was developed. It is based on surface energy transfer (SET) and an inner filter effect (IFE) between silicon nanoparticles and CoOOH nanoparticles as well as the redox reaction between vitamin C and CoOOH nanoparticles.

Amplification-free and direct fluorometric determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters.

A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557 nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a -TTA- loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/µL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost. Graphical Abstract A method was developed using chimeric DNA-templated silver nanoclusters to detect telomerase activity directly in cell extracts. The sensitivity of this new method outperforms the traditional TRAP assay, and without the need for amplification.

A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity.

Previous research has characterized the important role of aldehyde oxidases (AOX) in biotransformation of N-heterocyclic therapeutic drugs and environmental contaminants in mammals. Research pertaining to AOX activity in non-mammalian vertebrates, however, is scarce, despite its biological role as a potentially important metabolic pathway for xenobiotics. One of the limiting factors of research on AOX is that available photometric methods are relatively insensitive, limited in throughput, and prone to cross-reactivity from other enzymes. Therefore, this study aimed to develop a novel and improved fluorometric AOX assay. This assay is based on the conversion of the exogenous aldehyde substrate 4-(dimethyl)amino cinnamaldehyde to its corresponding fluorescent acid by AOX, and was evaluated using partially purified hepatic cytosol from rat, human, and rainbow trout. Purification of native cytosol by heat treatment and ammonium sulfate precipitation resulted in increased specific activity of AOX. Michaelis-Menten kinetic parameters (Kmand Vmax) were comparable to values previously generated by photometric methods. Furthermore, effects of the inhibitor hydralazine on AOX activity revealed half maximal inhibitory concentrations comparable to those generated using conventional methods. Product identity was confirmed by liquid chromatography and mass spectrometry. In summary, this study successfully developed a rapid and sensitive assay for determination of AOX activity in across different vertebrate species that is 4- to 10-fold more sensitive compared to conventional absorbance-based methods. It can be applied in environmental, toxicological, and pharmacological studies relating to identification of AOX substrates, as well as the induction of AOX expression through drugs and environmental contaminants.

A cascade amplification strategy of catalytic hairpin assembly and hybridization chain reaction for the sensitive fluorescent assay of the model protein carcinoembryonic antigen.

A cascade nucleic acid amplification strategy is presented for fluorometric aptamer based determination of the model protein carcinoembryonic antigen (CEA). Amplification is accomplished by combining catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). In this assay, a specially designed single-stranded DNA containing the aptamer sequence (AS) specific for CEA is hybridized with an inhibitor strand (IS) to form a double-stranded DNA (IS@AS). In the presence of CEA, it will recognize and bind to the AS strand which causes the release of IS. By introducing two DNA hairpins (H1 and H2; these containing complementary sequences) CHA will be activated via the hybridization reactions of H1 and H2. This is accompanying by the formation of a double-stranded DNA (H1-H2) and the release of CEA@AS. The liberated CEA@AS further drives successive recycling of the CHA, thereby generating further copies of H1-H2. The resultant H1-H2 hybrids act as primers and trigger HCR with the help of other two DNA hairpins (H3 and H4) containing G-rich toehold at the 5'-terminus and 3'-terminus of H3 and H4, respectively. The fluorescent probe N-methyl mesoporphyrin IX (NMM) is finally intercalated into the G-rich domains of the long DNA nanostructures due to formation of G-quadruplex structures. This generates a fluorescent signal (best measured at excitation/emission wavelengths of 399/610 nm) that increases with the concentration of target (CEA). This aptamer-based fluorescence assay is highly sensitive and has a linear range that covers the 1 pg·mL-1 to 2 ng·mL-1 CEA concentration range, with a 0.3 pg·mL-1 detection limit. Graphical abstract By integrating catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for effective signal enhancement, a novel cascade amplification strategy is presented to develop a sensitive and selective fluorescent method for the assay of the model protein carcinoembryonic antigen (CEA).

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.