Ultrasensitive miRNA Detection by AuNP-based 3D DNA Walker and Catalytic Hairpin Assembly (CHA) Cascade Amplification for Early Cancer Diagnosis.

By combining exquisitely designed hairpins with the catalytic hairpin assembly (CHA) to form tripedal DNA walkers driven by enzyme, we constructed a 3D DNA walker with accordingly complementary hairpins attached on gold nanoparticles (AuNPs) and sensitive fluorescence sensing system for the sensitive detection of target miRNA-21 (miR-21). The presence of miR-21 triggers the CHA among three hairpins (HP1, HP2, and HP3), which lead to the formation of the tripedal DNA walkers. For the walking trajectories, FAM-labeled hairpins (HP4) were attached to the surface of AuNPs, the fluorescence of which was initially quenched due to its close proximity to AuNPs. After the binding/cleaving/moving process of tripedal DNA walkers with HP4 driven by Exonuclease III (Exo III), a number of single-stranded DNAs (ssDNAs) will be released with FAM fluorescence recovered. Benefiting from the DNA walker and CHA cascade amplification, the proposed sensing strategy showed remarkable improvement in sensitivity with the LOD of 42 aM. Owing to the precise design of the system, this method exhibited excellent specificity to distinguish miR-21 from its single-, double-mismatched sequences and non-complementary sequences, showing great versatility and potential for the biological analysis and early disease diagnosis.

Multifunctional theranostic carbazole-based cyanine for real-time imaging of amyloid-ß and therapeutic treatment of multiple pathologies in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder characterized by the synaptic and neuronal loss, which results in cognitive impairment in particular learning and memory. Currently, AD is incurable and no single confirmative test can clinically be used to diagnose AD. In light of the complex and multifactorial nature of AD etiology, the development of multifunctional/multi-target drugs that act on multiple pathological pathways and mechanisms shows great therapeutic potential for intervention of this devastating disease. We report herein a multifunctional theranostic cyanine, SLCOOH, which serves not only as a highly sensitive fluorescent probe for real-time imaging of amyloid-ß (Aß) contents in different age groups of transgenic (Tg) AD mice but also as an effective therapeutic agent for early AD intervention via multiple pathological targets in the AD mouse model. Remarkably, treatment with SLCOOH gives rise to multiple therapeutic benefits, including the amelioration of cognitive decline, a reduction in Aß levels, a decrease in hyperphosphorylated tau proteins and tau depositions, and the alleviation of synaptic loss and dysfunctions in young triple Tg AD mice. Our results have demonstrated that in addition to superior Aß imaging capability, SLCOOH exhibits versatile and effective multiple modes of drug action, signifying outstanding therapeutic potential to treat early onset AD. Our work also paves the way for the development of effective Aß-targeted theranostic agents for AD.

Real-Time Detection and Visualization of Amyloid-ß Aggregates Induced by Hydrogen Peroxide in Cell and Mouse Models of Alzheimer's Disease.

Oxidative stress, caused by an imbalance between the production and the accumulation of reactive oxygen species (ROS), is a prominent cause of the neurotoxicity induced by aggregated amyloid-ß (Aß) in Alzheimer's disease (AD). Tools that can directly detect and monitor the presence and amount of Aß-induced ROS are still lacking. We report herein the first Aß-targeted ratiometric H2O2-responsive fluorescent probe for real-time detection and monitoring of the Aß-induced H2O2 level in cell and AD mouse models. The H2O2-responsive probe is constructed based on a methylamino-substituted quinolinium-based cyanine as the fluorescence moiety and a phenylboronate ester as the sensing reaction site. This sensing probe exhibits a large emission wavelength shift of ∼87 nm upon reacting with H2O2, a high binding selectivity for Aß, and a faster response toward H2O2 in the presence of Aß, concomitant with an enhanced fluorescence intensity, hence greatly boosting the sensitivity of in-situ H2O2 detection. This biocompatible and nontoxic probe is capable of ratiometrically detecting and imaging endogenous H2O2 induced by Aß in a neuronal cell model. Remarkably, this Aß-targeted H2O2-responsive probe is also able to detect, monitor, and differentiate different Aß-induced H2O2 levels in real time in different age groups of transgenic AD mice in which the cerebral H2O2 level increases age dependently concomitant with the plaque contents. Therefore, this smart probe can act as a powerful tool to diagnose high-risk subjects and diseased brains of AD and to further study the role of ROS in AD pathology.

Plasma amyloid-beta levels correlated with impaired hepatic functions: An adjuvant biomarker for the diagnosis of biliary atresia.

Background: Biliary atresia (BA) is an infantile fibro-obstructive cholestatic disease with poor prognosis. An early diagnosis and timely Kasai portoenterostomy (KPE) improve clinical outcomes. Aggregation of amyloid-beta (Aß) around hepatic bile ducts has been discovered as a factor for BA pathogenesis, yet whether plasma Aß levels correlate with hepatic dysfunctions and could be a biomarker for BA remains unknown. Method: Plasma samples of 11 BA and 24 controls were collected for liver function test, Aß40 and Aß42 measurement by enzyme-linked immunosorbent assay (ELISA). Pearson's chi-squared test or Mann-Whitney U test was performed to assess differences between groups. Correlation between Aß42/Aß40 and liver function parameters was performed using Pearson analysis. The area under the receiver-operative characteristic (ROC) curve (area under curve; AUC) was measured to evaluate the diagnostic power of Aß42/Aß40 for BA. Diagnostic enhancement was further evaluated by binary regression ROC analysis of Aß42/Aß40 combined with other hepatic function parameters. Results: Plasma Aß42/Aß40 was elevated in BA patients. Aß42 displayed a weak positive correlation with γ-glutamyl transpeptidase (GGT) (Pearson's correlation = 0.349), while there was no correlation for Aß40 with hepatic functions. Aß42/Aß40 was moderately correlated with GGT, total bile acid (TBA), direct bilirubin (DBIL) (Pearson's correlation = 0.533, 0.475, 0.480), and weakly correlated with total bilirubin (TBIL) (Pearson's correlation = 0.337). Aß42/Aß40 showed an acceptable predictive power for cholestasis [AUC = 0.746 (95% CI: 0.552-0.941), p < 0.05]. Diagnostic powers of Aß42/Aß40 together with hepatic function parameters for cholestasis were markedly improved compared to any indicator alone. Neither Aß42/Aß40 nor hepatic function parameters displayed sufficient power in discriminating BA from choledochal cysts (CC); however, combinations of Aß42/Aß40 + GGT along with any other hepatic function parameters could differentiate BA from CC-cholestasis (AUC = 1.000, p < 0.05) with a cut-off value as 0.02371, -0.28387, -0.34583, 0.06224, 0.01040, 0.06808, and 0.05898, respectively. Conclusion: Aß42/Aß40 is a good indicator for cholestasis, but alone is insufficient for a distinction of BA from non-BA. However, Aß42/Aß40 combined with GGT and one other hepatic function parameter displayed a high predictive power as a screening test for jaundiced neonates who are more likely to be BA, enabling them to early intraoperative cholangiography for BA confirmation and KPE to improve surgical outcomes. However, a multi-centers validation is needed before introduction into daily clinical practice.

2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.

Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in-situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal-organic framework nanosensor (Nano-DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis.

Correction: The morphology and surface charge-dependent cellular uptake efficiency of upconversion nanostructures revealed by single-particle optical microscopy.

[This corrects the article DOI: 10.1039/C8SC01828F.].

Theranostic F-SLOH mitigates Alzheimer's disease pathology involving TFEB and ameliorates cognitive functions in Alzheimer's disease models.

Accumulation of amyloid-ß (Aß) oligomers and phosphorylated Tau aggregates are crucial pathological events or factors that cause progressive neuronal loss, and cognitive impairments in Alzheimer's disease (AD). Current medications for AD have failed to halt, much less reverse this neurodegenerative disorder; therefore, there is an urgent need for the development of effective and safe drugs for AD therapy. In the present study, the in vivo therapeutic efficacy of an Aß-oligomer-targeted fluorescent probe, F-SLOH, was extensively investigated in 5XFAD and 3XTg-AD mouse models. We have shown that F-SLOH exhibits an efficient inhibitory activity against Aß aggregation in vivo, and acts as an effective theranostic agent for the treatment of multiple neuropathological changes in AD mouse models. F-SLOH has been found to significantly reduce not only the levels of Aß oligomers, Tau aggregates and plaques but also the levels of amyloid precursor protein (APP) and its metabolites via autophagy lysosomal degradation pathway (ALP) in the brains of 5XFAD and 3XTg-AD mice. It also reduces astrocyte activation and microgliosis ultimately alleviating neuro-inflammation. Furthermore, F-SLOH mitigates hyperphosphorylated Tau aggregates, synaptic deficits and ameliorates synaptic memory function, and cognitive impairment in AD mouse models. The mechanistic studies have shown that F-SLOH promotes the clearance of C-terminal fragment 15 (CTF15) of APP and Paired helical filaments of Tau (PHF1) in stable cell models via the activation of transcription factor EB (TFEB). Moreover, F-SLOH promotes ALP and lysosomal biogenesis for the clearance of soluble, insoluble Aß, and phospho Tau. Our results unambiguously reveal effective etiological capabilities of theranostic F-SLOH to target and intervene multiple neuropathological changes in AD mouse models. Therefore, F-SLOH demonstrates tremendous therapeutic potential for treating AD in its early stage.

Saccharide-Functionalized Poly(Zn-salphen)-alt-(m- and p-phenyleneethynylene)s as Dynamic Helical Metallopolymers.

The study of metallopolymers with controllable helical sense remains in its infancy. We report arabinose-functionalized (Zn-salphen)-based conjugated polymers that display mirror-image circular dichroism spectra for L- and D-sugar sidechains respectively, signifying ordered (helical) coiling of the polymer backbone with opposite screw-sense preferences. The observation of different spectroscopic behavior and Cotton effects for a variety of solvents (in a reversible manner) and temperatures, ascribed to changes in the extent of intrachain (Zn⋅⋅⋅O(salphen) and π-stacking) interactions between Zn-salphen moieties, thus indicate the flexible, responsive and dynamic nature of the folded helical conformation in these systems. An application study signifying that activity can be governed by the structure and helical sense of the polymer is described.

Multimodal Theranostic Cyanine-Conjugated Gadolinium(III) Complex for In Vivo Imaging of Amyloid-ß in an Alzheimer's Disease Mouse Model.

Despite the wide use of magnetic resonance imaging (MRI) as a clinical diagnostic tool, there are still no clinically approved MRI contrast agents that can be applied for cerebral Alzheimer's disease (AD) biomarker imaging. We report here the design and development of the first amyloid-ß (Aß)-targeted, blood-brain barrier (BBB) penetrable theranostic Gd(DOTA)-cyanine dyad, which was synthesized by the conjugation of Gd(DOTA) complex and carbazole-based cyanine dye by the copper(I)-catalyzed azide-alkyne cycloaddition click reaction for imaging of Aß in vivo and ex vivo in AD mouse models. This dyad, as a multimodal probe, possesses desirable multifunctional properties, including good biocompatibility, low cytotoxicity, high Aß selectivity, strong fluorescence enhancement upon binding with Aß species, good paramagnetic properties, high stability, good BBB penetrability, and fast elimination from the mouse. The longitudinal relaxivity (r1) of the dyad was found to be 4.42 mM-1 s-1 at 3 T, suggesting it to be promising as a T1-weighted MRI contrast agent. The probe has been successfully demonstrated to be able to be applied for one- and two-photon excited fluorescence and magnetic resonance (MR) imaging of Aß in transgenic mouse models of AD. In addition, it can inhibit Aß aggregation, protect against toxicity induced by Aß, and suppress Aß-induced reactive oxygen species (ROS) production. Our results demonstrate the highly promising theranostic capability of the dyad for diagnosis and therapy of AD and extraordinary potential for MRI of Aß in humans.

Amyloid-ß oligomer targeted theranostic probes for in vivo NIR imaging and inhibition of self-aggregation and amyloid-ß induced ROS generation.

To enable the early detection and intervention of Alzheimer's disease (AD), it is highly desirable to develop novel theranostic agents for simultaneous detection of toxic and pathogenic amyloid-ß (Aß) oligomers in vivo and attenuation of Aß-induced toxicity. Herein, we report a new series of oligomeric Aß targeted near infrared (NIR) emissive dibutylnaphthylamine-based cyanine probes for in vivo and ex vivo imaging of Aß in AD mouse model. These new fluorophores exhibited strong solvatochromism and a large bathochromic shift of the emission spectrum upon binding with Aß species, giving rise to advantageous NIR emission. Besides, they showed an intriguingly stronger fluorescence enhancement upon interacting with Aß oligomers and monomers, and binding affinity toward Aß oligomers and monomers than Aß fibrils, suggesting they were selective to Aß oligomers and monomers. In addition to low toxicity, one of the fluorophores, DBAN-SLM, showed remarkably effective inhibitory effect on Aß aggregation, significant neuroprotection effect against the Aß-induced toxicities, and suppression on Aß-induced reactive oxygen species (ROS) generation. Because of excellent blood-brain barrier (BBB) permeability, good biocompatibility and stability, high specificity towards Aß oligomers as well as strong turn-on fluorescence upon Aß binding, DBAN-SLM was successfully applied for in vivo and ex vivo imaging of Aß in AD mouse model, signifying its great promise as a useful theranostic agent for the early diagnosis and therapy of AD. Our results also demonstrated for the first time that the dibutyl-2-naphthylamine moiety is a useful and effective structural building block to promote the targeting capability of oligomeric Aß.

Rapid and ultrasensitive detection of food contaminants using surface-enhanced Raman spectroscopy-based methods.

With the globalization of food and its complicated networking system, a wide range of food contaminants is introduced into the food system which may happen accidentally, intentionally, or naturally. This situation has made food safety a critical global concern nowadays and urged the need for effective technologies capable of dealing with the detection of food contaminants as efficiently as possible. Hence, Surface-enhanced Raman spectroscopy (SERS) has been taken as one of the primary choices for this case, due to its extremely high sensitivity, rapidity, and fingerprinting interpretation capabilities which account for its competency to detect a molecule up to a single level. Here in this paper, we present a comprehensive review of various SERS-based novel approaches applied for direct and indirect detection of single and multiple chemical and microbial contaminants in food, food products as well as water. The aim of this paper is to arouse the interest of researchers by addressing recent SERS-based, novel achievements and developments related to the investigation of hazardous chemical and microbial contaminants in edible foods and water. The target chemical and microbial contaminants are antibiotics, pesticides, food adulterants, Toxins, bacteria, and viruses. In this paper, different aspects of SERS-based reports have been addressed including synthesis and use of various forms of SERS nanostructures for the detection of a specific analyte, the coupling of SERS with other analytical tools such as chromatographic methods, combining analyte capture and recognition strategies such as molecularly imprinted polymers and aptasensor as well as using multivariate statistical analyses such as principal component analysis (PCA)to distinguish between results. In addition, we also report some strengths and limitations of SERS as well as future viewpoints concerning its application in food safety.

Natural protein-templated fluorescent gold nanoclusters: Syntheses and applications.

For the past decades, the synthesis of metal nanoclusters has been a great interest for research, for their unique physicochemical properties and great contributions to the catalytic, electrical and biomedical applications. Protein-templated gold nanoclusters (AuNCs) is a kind of fluorescent nanomaterials with good solubility, excellent stability, biocompatibility, decent quantum yields and active groups (-COOH, -NH2) for facilitating modifications. Natural proteins are easily available, commercially affordable, diverse and multitudinous in animals, plants and foods, which provide a template pool for the exploration of AuNCs. This is one of the few reviews of specifically focusing on the natural protein-templated fluorescent AuNCs. The syntheses, properties and applications of different AuNCs were enumerated. Prospects were given on utilizing structure-modified proteins, bioactive enzymes, antibodies which should endow the AuNCs more favourable fluorescence performances and functional characteristics. The applications of AuNCs in analytical, biomedical and food sciences would be further heightened.

A sandwich-type surface-enhanced Raman scattering sensor using dual aptamers and gold nanoparticles for the detection of tumor extracellular vesicles.

A sandwich-type surface-enhanced Raman scattering (SERS) sensor using dual aptamers and gold-enhanced Raman signal probes has been successfully constructed for the detection of tumor-derived extracellular vesicles. The simple and sensitive sensor has the capability to detect tumor extracellular vesicles in 10-fold diluted human serum samples.

Deep Red Blinking Fluorophore for Nanoscopic Imaging and Inhibition of ß-Amyloid Peptide Fibrillation.

Deposition and aggregation of ß-amyloid (Aß) peptides are demonstrated to be closely related to the pathogenesis of Alzheimer's disease (AD). Development of functional molecules capable of visualizing Aß1-40 aggregates with nanoscale resolution and even modulating Aß assembly has attracted great attention recently. In this work, we use monocyanine fluorophore as the lead structure to develop a set of deep red carbazole-based cyanine molecules, which can specifically bind with Aß1-40 fibril via electrostatic and van der Waals interactions. Spectroscopic and microscopic characterizations demonstrate that one of these fluorophores, (E)-1-(2-(2-methoxyethoxy)ethyl)-4-(2-(9-methyl-9H-carbazol-3-yl)vinyl) quinolinium iodide (me-slg) can bind to Aß1-40 aggregates with strong fluorescence enhancement. The photophysical properties of me-slg at the single-molecule level, including low "on/off" duty cycle, high photon output, and sufficient switching cycles, enable real-time nanoscopic imaging of Aß1-40 aggregates. Morphology-dependent toxic effect of Aß1-40 aggregates toward PC12 cells is unveiled from in situ nanoscopic fluorescence imaging. In addition, me-slg displays a strong inhibitory effect on Aß1-40 fibrillation in a low inhibitor-protein ratio (e.g., I:P = 0.2). A noticeably reduced cytotoxic effect of Aß1-40 after the addition of me-slg is also confirmed. These results afford promising applications in the design of a nanoscopic imaging probe for amyloid fibril as well as the development of inhibitors to modulate the fibrillation process.

A fluorometric assay of thrombin using magnetic nanoparticles and enzyme-free hybridization chain reaction.

A fluorescence method based on functionalized magnetic nanoparticles (FMNPs) and hybridization chain reaction (HCR) is developed for the enzyme-free amplified determination of thrombin. In the proposed design, aptamer against thrombin was hybridized with the capture DNA-modified magnetic nanoparticles to yield the FMNPs. In the presence of thrombin, aptamers are released due to the specific and high-affinity binding between thrombin and its aptamer. The exposed capture DNA subsequently hybridized with the partial sequence of helper DNA, and the vacant sequence of helper DNA further hybridized with HCR products which is pre-formed by the alternate hybridization of single-stranded DNAs (H1 and H2). The immobilized HCR products were then labeled with YOYO-1 for fluorescence measurement. Fluorescence signal intensity of labeled YOYO-1 was measured at an emission wavelength of 519 nm (excitation under 488 nm) and used for calibration. By taking advantage of HCR amplification, this direct assay strategy showed a linear response in the 20- to 200-pM concentration range, and the limit of detection is 9.2 pM which is about 3-orders of magnitude lower than the serum thrombin concentration (10 nM) that triggers blood clotting. This developed method can efficiently differentiate the target protein from a protein matrix, and it is verified by determination of thrombin in spiked serum samples with recoveries in the range of 94.5-103.3%. Graphical abstract A fluorometry method for thrombin detection using magnetic nanoparticles and enzyme-free hybridization chain reaction.

A simple, sensitive and non-enzymatic signal amplification strategy driven by seesaw gate.

Herein, a simple enzyme-free method based on the seesaw-gate-driven isothermal signal amplification strategy was developed for nucleic acid detection. In this method, a partially complementary double-stranded beacon was designed, after the addition of ssDNA or RNA of target sequence, the fluorescence signal was restored through a toehold-mediated strand displacement process, followed by a seesaw-like reaction with the aid of an auxiliary strand with the same length of the toehold domain. Liberation of the target would initiate the next round of seesaw reaction to achieve recycling amplification of the fluorescence signal. The method has the advantages of simple sequence design and free of any enzyme, which can realize rapid detection of the target at 25 °C with a detection limit of 9.8 pM for DNA and 83 pM for RNA. The potential applicability of the proposed method was also demonstrated, indicating that it can provide a fundamental strategy for the development of nucleic acid sensors.

A fast detection of peroxynitrite in living cells.

Peroxynitrite (ONOO-) plays a crucial role in the regulation of diverse pathophysiological processes, and high level of ONOO- is profound association with numerous diseases. Herein, we developed an anthraquinone-based fluorescent probe L for ONOO- determination by a new recognition mechanism: amido oxidized nitroso-group by ONOO-. Probe L with amine-based recognition receptor is more selective to ONOO- than other reactive oxygen species, including H2O2 and ClO-. Furthermore, ONOO- could be rapidly detected by probe L with a Limit of Detection of 13 nM. More importantly, L could be used to monitor intracellular ONOO- in SMMC-7721 cells.

DNA-Hairpin-Templated Silver Nanoclusters: A Study on Stem Sequence.

DNA hairpins are widely used to synthesize silver nanoclusters (AgNCs) with excellent optical properties due to their specific secondary structure. Hairpin-AgNCs have been popularly employed for sensoring applications, while no systematic study has been done about the effect of stem sequence on the fluorescence property of hairpin-AgNCs. In this presented work, the synthesizing conditions of hairpin-AgNCs were fully examined first. Then, the effect of percentage content and distribution of GC base pairs as well as stem length on the fluorescence property of hairpin-AgNCs were studied. Intriguing phenomena were observed and basic conclusions were drawn, which would be helpful to understand the hairpin-AgNCs comprehensively and instructional for the applications using hairpin-AgNC probes.

Cognitive improvement and synaptic deficit attenuation by a multifunctional carbazole-based cyanine in AD mice model through regulation of Ca2+/CaMKII/CREB signaling pathway.

Accumulation of ß-amyloid (Aß) peptide and hyperphosphorylated tau in the brain is one of the pathological characteristics of Alzheimer's disease (AD) and attractive therapeutic targets in its treatment. In the present study, the cognitive ability of 4-month-old 3 × Tg-AD mice significantly improved after 40 days treatment with intraperitoneal injection of 2.25 mg/kg of SLOH, which is a multifunctional carbazole-based cyanine fluorophore. It reduced Aß deposition, tau levels and its hyperphosphorylation by modulating AKT and promoting protein phosphatase 2A activity in the brain as well as in the primary neurons of 3 × Tg-AD mice. Moreover, SLOH attenuated synaptic deficit both in vitro and in vivo by regulating the Ca2+/CaMKII/CREB signaling pathway. These findings strongly suggest that SLOH owns a high therapeutic potential to treat early onset AD.