Direct and sensitive detection of circulating miRNA in human serum by ligase-mediated amplification.

MicroRNAs (miRNA) involve in regulating different physiological processes whose dysregulation is associated with a wide range of diseases including cancers, diabetes and cardiovascular problems. Herein, we report a direct, sensitive and highly selective detection assay for circulating microRNA (miRNA). This detection strategy employs magnetic nanoparticles as the reaction platform which can not only allow online pre-concentration and selective separation but also integrates ligation reaction with amplification to enhance the sensitivity of the detection assay. With the presence of the target miRNA, the locked nucleic acid (LNA)-modified molecular beacon (MB) opens up, exposing the binding sites at two ends. The 3'- and 5'-end of the MB responsible for the attachment onto the magnetic nanoparticles, and reporting probe for the attachment of the pair of amplification probes respectively. The ligase ligate RNA to DNA enhance the amplification efficiency. Upon labelled with intercalating fluorophores (YOYO-1) on the hybrids, the quantification of the target miRNA was determined by measuring the fluorescence intensity. A detection limit of 314 fM was achieved with trace amount of sample consumption (~20 µL). As a proof of concept, miRNA-149 was chosen as the target miRNA. This assay is capable of discriminating single-base and reliably quantifying circulating miRNA-149 in both healthy and cancer patient's serums. The result obtained was comparable with that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), suggesting that this direct and sensitive assay can be served as a promising, non-invasive tool for early diagnosis of breast cancer and colorectal cancer.

Amyloid-ß Aggregation Inhibitory and Neuroprotective Effects of Xanthohumol and its Derivatives for Alzheimer's Diseases.

BACKGROUND: Xanthohumol has been reported to have cytoprotection through activation of Nrf2-ARE signaling pathway and; it has capability of scavenging free radicals, suggesting its potential for the prevention of neurodegeneration. However, the bio-incompatibility and blood-brain barrier impermeability of xanthohumol hindered its in vivo efficacy potential for treating Alzheimer's disease (AD). OBJECTIVE: We designed and prepared a series of xanthohumol derivatives to enhance the desirable physical, biological and pharmacological properties in particular the blood-brain barrier permeability for intervention of AD. METHODS: We designed and synthesized a novel series of 9 xanthohumol derivatives. Their inhibitory effect on amyloid-ß (1-42), Aß1-42, oligomerization and fibrillation as well as neuroprotection against amyloid-ß induced toxicities, were explored. RESULTS: Among the 9 xanthohumol derivatives, some of them exhibited a moderate to high inhibitory effect on Aß1-42 oligomerization and fibrillation. They were biocompatible and neuroprotective to the SH-SY5Y cells by reducing the ROS generation and calcium uploading that were induced by the amyloid- ß. Importantly, two of the derivatives were found to be blood-brain barrier permeable showing promising potential for AD treatment. CONCLUSION: Two derivatives have been identified to be biocompatible, non-toxic, neuroprotective against Aß-induced toxicities and blood-brain barrier permeable highlighting their promising potential as AD drug candidates for future clinical use.

Total internal reflection-based single-vesicle in situ quantitative and stoichiometric analysis of tumor-derived exosomal microRNAs for diagnosis and treatment monitoring.

Purpose: Exosomes (EXs) have been increasingly recognized as natural nanoscale vehicles for microRNA (miRNA)-based cell-cell communication and an ideal source of miRNA biomarkers in bodily fluids. Current methods allow bulk analysis of the miRNA contents of EXs, but these approaches are not suitable for the in situ stoichiometry of exosomal miRNAs and fail to reveal phenotypic heterogeneity at the single-vesicle level. This study aimed to develop a single vesicle-based, mild, precise, but versatile method for the in situ quantitative and stoichiometric analysis of exosomal miRNAs. Methods: A total internal reflection fluorescence (TIRF)-based single-vesicle imaging assay was developed for direct visualization and quantification of the single-vesicles of EXs and their miRNA contents in serum microsamples. The assay uses co-delivery of inactive split DNAzymes and fluorescence-quenched substrates into nanosized EXs treated with streptolysin O to produce a target miRNA-activated catalytic cleavage reaction that amplifies the readout of fluorescence signal. We perform the in situ quantitative and stoichiometric analysis of serum exosomal hsa-miRNA-21 (miR-21), a common cancer biomarker, by using the developed TIRF imaging assay. Results: The TIRF imaging assay for serum exosomal miR-21 can distinguish cancer patients from healthy subjects with better performance than conventional real-time polymerase chain reaction (PCR) assay. The exosomal miR-21 level in serum is also informative for monitoring tumor progression and responses to treatment. Moreover, the TIRF assays can readily determine the precise stoichiometry of target exosomal miRNA contents in situ by delivering molecular beacon (MB) probes into EXs. Conclusions: The created TIRF imaging platform shows high applicability to serve as a universal and useful tool for the single-vesicle in situ quantitative and stoichiometric analysis of other disease-associated exosomal miRNAs markers and provide valuable insight into the physiological relevance of EX-mediated miRNA communication.

Highly sensitive quantification of Alzheimer's disease biomarkers by aptamer-assisted amplification.

Alzheimer's disease (AD), a chronic neurodegenerative disease associated with the loss of neurons in the brain, is the most pervasive type of dementia; 47 million people are affected, and the number is expected to increase to more than 131 million by 2050, according to Alzheimer's Disease International. Both early diagnosis and continuous monitoring are crucial for early intervention, symptomatic treatment, monitoring of the efficacy of intervention and improved patient function. Beta-amyloid peptide, tau, and phosphorylated tau are useful for screening and diagnosis; meanwhile, simultaneous assessment of multiple biomarkers is of paramount importance for accurate disease diagnosis. Methods: Herein, we report a direct, inexpensive and ultrasensitive aptamer-based multiplex assay for the quantification of trace amounts of AD biomarkers in both human serum and cerebrospinal fluid (CSF) samples. In this newly developed assay, molecular recognition of an antibody-aptamer pair provides high specificity in target detection, and the use of a DNA amplification strategy affords high sensitivity, allowing quantification of AD biomarkers in both biological fluids in 1.5 h with only a diminutive amount of the sample consumed. A tailor-made turn-on fluorophore, namely, SPOH, was employed to label the antibody-aptamer hybrids and provide a strong fluorescence signal, which was then detected with a total internal reflection fluorescence microscopy electron-multiplying charge-coupled device (TIRFM-EMCCD) imaging system. The simultaneous detection of biomarkers was achieved by a direct shape-coded method in which the nanoplatforms can be distinguished from one another by their morphologies. Results: This assay demonstrated a lower detection limit (in the femtomolar range) for AD biomarkers than the previously reported antibody-antibody method. Conclusion: The developed assay holds tremendous clinical potential for early diagnosis of AD and monitoring of its progression.

Molecular-Recognition-Based DNA Nanodevices for Enhancing the Direct Visualization and Quantification of Single Vesicles of Tumor Exosomes in Plasma Microsamples.

Tumor exosomes (Exo) are presumed to expedite both the growth and metastasis of tumors by actively participating in nearly all aspects of cancer development. Tumor-derived Exos are thus proposed as a resource for diagnostic biomarkers in bodily fluids. However, most Exo assays require large samples and are time-consuming, complicated, and costly, and thus unsuited for practical applications. Herein, we show an ultrasensitive assay that can directly visualize and quantify tumor Exos in plasma microsamples (1 µL) at the single-vesicle level. The assay uses the specific binding of activatable aptamer probes (AAP) to target Exos captured by Exo-specific antibodies on the surface of a flow cell to produce activated fluorescence. Furthermore, the bound AAP triggers in situ assembly of a DNA nanodevice with enhanced fluorescence that improves the Exo-detection sensitivity. By identifying tyrosine-protein-kinase-like 7 (PTK7), a total-internal-reflection-fluorescence (TIRF) assay for PTK7-Exo distinguishes target tumors from control subjects. This assay is also informative in monitoring tumor progression and early responses to therapy. The developed assay can be readily adapted for diagnosis and monitoring of other disease-associated Exo biomarkers.

Silica nanoparticles induce neurodegeneration-like changes in behavior, neuropathology, and affect synapse through MAPK activation.

BACKGROUND: Silica nanoparticles (SiO2-NPs) are naturally enriched and broadly utilized in the manufacturing industry. While previous studies have demonstrated toxicity in neuronal cell lines after SiO2-NPs exposure, the role of SiO2-NPs in neurodegeneration is largely unknown. Here, we evaluated the effects of SiO2-NPs-exposure on behavior, neuropathology, and synapse in young adult mice and primary cortical neuron cultures. RESULTS: Male C57BL/6 N mice (3 months old) were exposed to either vehicle (sterile PBS) or fluorescein isothiocyanate (FITC)-tagged SiO2-NPs (NP) using intranasal instillation. Behavioral tests were performed after 1 and 2 months of exposure. We observed decreased social activity at both time points as well as anxiety and cognitive impairment after 2 months in the NP-exposed mice. NP deposition was primarily detected in the medial prefrontal cortex and the hippocampus. Neurodegeneration-like pathological changes, including reduced Nissl staining, increased tau phosphorylation, and neuroinflammation, were also present in the brains of NP-exposed mice. Furthermore, we observed NP-induced impairment in exocytosis along with decreased synapsin I and increased synaptophysin expression in the synaptosome fractions isolated from the frontal cortex as well as primary neuronal cultures. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were also activated in the frontal cortex of NP-exposed mice. Moreover, inhibition of ERK activation prevented NP-mediated changes in exocytosis in cultured neurons, highlighting a key role in the changes induced by NP exposure. CONCLUSIONS: Intranasal instillation of SiO2-NPs results in mood dysfunction and cognitive impairment in young adult mice and causes neurodegeneration-like pathology and synaptic changes via ERK activation.

Effective Theranostic Cyanine for Imaging of Amyloid Species in Vivo and Cognitive Improvements in Mouse Model.

We report herein an investigation of carbazole-based cyanine, (E)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)-vinyl)-1-methyl-quinolin-1-iumiodide (SLM), as an effective theranostic agent for Alzheimer's disease (AD). This cyanine exhibited desirable multifunctional and biological properties, including amyloid-ß (Aß)-oligomerization inhibition, blood-brain barrier permeability, low neurotoxicity, neuroprotective effect against Aß-induced toxicities, high selectivity and strong binding interactions with Aß peptide/species, good biostability, as well as strong fluorescence enhancement upon binding to Aß species for diagnosis and therapy of AD. This cyanine has been successfully applied to perform near-infrared in vivo imaging of Aß species in transgenic AD mouse model. The triple transgenic AD mice intraperitoneally treated with SLM showed significant recovery of cognitive deficits. Furthermore, those SLM-treated mice exhibited a substantial decrease in both of oligomeric Aß contents and tau proteins in their brain, which was attributed to the induction of autophagic flux. These findings demonstrated for the first time that SLM is an effective theranostic agent with in vivo efficacy for diagnosis and treatment of AD in mouse models.

Dual-Modal NIR-Fluorophore Conjugated Magnetic Nanoparticle for Imaging Amyloid-ß Species In Vivo.

Senile plaques, the extracellular deposit of amyloid-ß (Aß) peptides, are one of the neuropathological hallmarks found in Alzheimer's disease (AD) brain. The current method of brain imaging of amyloid plaques based on positron emission tomography (PET) is expensive and invasive with low spatial resolution. Thus, the development of sensitive and nonradiative amyloid-ß (Aß)-specific contrast agents is highly important and beneficial to achieve early AD detection, monitor the disease progression, and evaluate the effectiveness of potential AD drugs. Here a neuroprotective dual-modal nanoprobe developed by integrating highly Aß-specific and turn-on fluorescence cyanine sensors with superparamagnetic iron oxide nanoparticles as an effective near-infrared imaging (NIRI)/magnetic resonance imaging (MRI) contrast agent for imaging of Aß species in vivo is reported. This Aß-specific probe is found not only nontoxic and noninvasive, but also highly blood brain barrier permeable. It also shows a potent neuroprotective effect against Aß-induced toxicities. This nanoprobe is successfully applied for in vivo fluorescence imaging with high sensitivity and selectivity to Aß species, and MRI with high spatial resolution in an APP/PS1 transgenic mice model. Its potential as a powerful in vivo dual-modal imaging tool for early detection and diagnosis of AD in humans is affirmed.

Fluoro-substituted cyanine for reliable in vivo labelling of amyloid-ß oligomers and neuroprotection against amyloid-ß induced toxicity.

Alzheimer's disease (AD) is the most prevalent but still incurable neurodegenerative form of dementia. Early diagnosis and intervention are crucial for delaying the onset and progression of the disease. We herein report a novel fluoro-substituted cyanine, F-SLOH, which exhibits good Aß oligomer selectivity with a high binding affinity, attributed to the synergistic effect of strong π-π stacking and intermolecular CH···O and CH···F interactions. The selectivity towards the Aß oligomers in the brain was ascertained by in vitro labelling on tissue sections and in vivo labelling through the systemic administration of F-SLOH in 7 month APP/PS1 double transgenic (Tg) and APP/PS1/Tau triple Tg mouse models. F-SLOH also shows remarkably effective inhibition on Aß aggregation and highly desirable neuroprotective effects against Aß-induced toxicities, including the inhibition of ROS production and Ca2+ influx. Its excellent blood-brain barrier (BBB) penetrability and low bio-toxicity further support its tremendous potential as a novel theranostic agent for both early diagnosis and therapy of AD.

Ultra-sensitive detection of protein biomarkers for diagnosis of Alzheimer's disease.

Beta amyloid peptide, tau, and phosphorylated tau are well recognized as promising biomarkers for the diagnosis of Alzheimer's disease (AD). In this work, we developed a direct, versatile, and ultrasensitive multiplex assay for the quantification of trace amounts of these protein biomarkers for AD in different types of biological fluids including cerebrospinal fluid, serum, saliva, and urine. The detection assay is based on the immunoreaction between the target proteins and their corresponding pair of antibodies followed by fluorescence labelling with a newly developed indolium-based turn-on fluorophore, namely SIM. SIM was tailor-made as a reporter to provide a high signal-to-noise ratio for the detection assay. An exceptionally low limit of detection down to the femto-molar level was achieved in this assay with minute consumption of the sample. This versatile detection assay is capable of reliably quantifying not only the target proteins simultaneously from a CSF sample in an hour but also trace amounts of protein biomarkers in saliva and urine. This assay has a high potential to serve as a practical tool for the diagnosis of AD.

A theranostic agent for in vivo near-infrared imaging of ß-amyloid species and inhibition of ß-amyloid aggregation.

Amyloid-ß (Aß) peptide as one of the main components of senile plaques is closely related to the onset and progression of incurable Alzheimer's disease (AD). Numerous efforts have been devoted to develop probes for Aß species/plaque imaging for AD diagnostics and to develop aggregation inhibitors preventing formation of toxic soluble oligomeric Aß for therapeutics. Herein, for the first time, a series of novel charged molecules, which can simultaneously perform near infra-red in vivo imaging of Aß species/plaques in animal model and inhibition of self-aggregation of Aß monomer from forming toxic oligomers, are reported. Among them, DBA-SLOH showed excellent blood-brain barrier (BBB) permeability and biocompatibility due to the incorporation of lipophilic alkyl chains with moderate length into the charged skeleton. Importantly, DBA-SLOH was found to have a high binding affinity toward Aß species exhibiting a dramatic fluorescence enhancement upon interacting with Aß species. Despite a weaker binding with Aß monomers as compared to Aß aggregates, DBA-SLOH could effectively prevent the Aß1-40 and Aß1-42 peptides from self-aggregation and forming toxic oligomers. This multifunctional fluorescent molecule shows promising potential as a theranostic agent for the diagnosis and therapy of AD.

Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform.

A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. To afford a high signal-to-noise ratio, three turn-on fluorophores with unique optical properties have been designed and synthesized to label the target antigens. The developed assay has achieved a remarkable LOD down to the femto-molar regime without sample pre-treatment. This versatile assay can efficiently differentiate the target antigen from a protein matrix and simultaneously quantify multiple cancer-associated antigens, for instance, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) using only 6 µL of serum sample in an hour. This novel system has a high applicability to serve as a universal and useful tool for early disease diagnostics.

Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes.

We report herein the application of kinetically inert luminescent iridium(III) complexes as dual inhibitors and probes of beta-amyloid fibrillogenesis. These iridium(III) complexes inhibited Aß1-40 peptide aggregation in vitro, and protected against Aß-induced cytotoxicity in neuronal cells. Furthermore, the complexes differentiated between the aggregated and unaggregated forms of Aß1-40 peptide on the basis of their emission response.

Inhibition of ß-amyloid Aggregation By Albiflorin, Aloeemodin And Neohesperidin And Their Neuroprotective Effect On Primary Hippocampal Cells Against ß-amyloid Induced Toxicity.

Being one of the hallmarks of Alzheimer's disease, ß-amyloid (Aß) aggregates induce complicated neurotoxicity. Evidences show that the underlying mechanism of neurotoxicity involves a glutamate receptor subtype, N-methyl-D-aspartate (NMDA) receptor, an increase in intracellular calcium(II) ion loading as well as an elevation in oxidation stress. In this work, among the 35 chemical components of Chinese herbal medicines (CHMs) being screened for inhibitors of Aß aggregation, four of them, namely albiflorin, aloeemodin, neohesperidin and physcion, were found for the first time to exhibit a potent inhibitory effect on Aß(1-40) and Aß(1-42) aggregation. Their neuroprotective capability on primary hippocampal neuronal cells was also investigated by MTT assay, ROS assay and intracellular calcium(II) ion concentration measurement. It was interesting to find that physcion was rather toxic to neuronal cells while albiflorin, aloeemodin and neohesperidin reduced the toxicity and ROS induced by both monomeric and oligomeric Aß species. In addition, albiflorin was particularly powerful in maintaining the intracellular Ca(2+) concentration.

Direct quantification of circulating miRNAs in different stages of nasopharyngeal cancerous serum samples in single molecule level with total internal reflection fluorescence microscopy.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate human gene expression at the post-transcriptional level. Growing evidence indicates that the expression profile of miRNAs is highly correlated with the occurrence of human diseases including cancers. Playing important roles in complex gene regulation processes, the aberrant expression pattern of various miRNAs is implicated in different types and even stages of cancer. Besides localizing in cells, many of these miRNAs are found circulating around the body in a wide variety of fluids such as urine, serum and saliva. Surprisingly, these extracellular circulating miRNAs are highly stable and resistant to degradation, and therefore, are considered as promising biomarkers for early cancer diagnostic via noninvasive extraction from body fluids. Unfortunately, the abundance of these small RNAs is ultralow in the body fluids, making it challenging to quantify them in complex sample matrixes. Establishing a sensitive, specific yet simple assay for an accurate quantification of circulating miRNAs is therefore desirable. Our group previously reported a sensitive and specific detection assay of miRNAs in single molecule level with the aid of total internal reflection fluorescence microscopy. In this work, we advanced the assay to differentiate the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, a locked nucleic acid-modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 µL of serum and the whole assay can be completed in 1 h. The experimental results agreed well with those previously reported whereas the quantity of miR-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising noninvasive detection tool for early NPC diagnosis, monitoring and staging.

Self-assembling protein platform for direct quantification of circulating microRNAs in serum with total internal reflection fluorescence microscopy.

MicroRNA (miRNA) has recently emerged as a new and important class of cellular regulators. Strong evidences showed that aberrant expression of miRNA is associated with a broad spectrum of human diseases, such as cancer, diabetes, cardiovascular and psychological disorders. However, the short length and low abundance of miRNA place great challenges for conventional techniques in the miRNA quantification and expression profiling. Here, we report a direct, specific and highly sensitive yet simple detection assay for miRNA without sample amplification. A self-assembled protein nanofibril acted as an online pre-concentrating sensor to detect the target miRNA. Locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient's serums was also demonstrated.

Effect of surface-functionalized nanoparticles on the elongation phase of beta-amyloid (1-40) fibrillogenesis.

The influence of nanoparticles of various sizes and surface functionalities on the self-assembling fibrillogenesis of beta-amyloid (1-40) peptide was investigated. Functionalized nanoparticles including quantum dots and gold nanoparticles were co-incubated with monomeric Aß(1-40) peptides under seed-mediated growth method to study their influences on the elongation phase of the fibrillogenesis. It is observed that charge-to-surface area ratio of the nanoparticles and the functional moiety and electrostatic charges of the conjugated ligands on the particle surfaces took crucial regulatory role in the Aß(1-40) fibrillogenesis.