A simple, rapid and sensitive sandwich immunoassay based on poly(N-isopropylacrylamide) for the detection of alpha-fetoprotein.

Alpha-fetoprotein (AFP), as a tumor marker, plays a vital role in the diagnosis of liver cancer. In this work, a novel sandwich immunoassay based on a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), was developed for the detection of AFP. This immunoassay could realize one-step rapid reaction within 1 h, and facilitate the separation of the target molecules by incorporating PNIPAM. In this method, a conjugate of PNIPAM and capture antibody (Ab1) was successfully synthesized as a capture probe and the synthetic method of PNIPAM-Ab1 was simple, while the detection antibody (Ab2) was labeled with fluorescein isothiocyanate (FITC) to form a fluorescent detection probe. By employing a sandwich immunoassay, the method achieved quantitative determination of AFP, exhibiting a wide linear range from 5 ng/mL to 200 ng/mL and a low detection limit of 2.44 ng/mL. Furthermore, it was successfully applied to the analysis of spiked human serum samples and the screening of patients with hepatic diseases in clinical samples, indicating its potential application prospect in the diagnosis of liver cancer.

Meg3 Induces EMT and Invasion of Glioma Cells via Autophagy.

BACKGROUND: Glioma is one of the most common malignant tumors. Glioblastoma (grade IV) is considered the most malignant form of human brain tumors. Maternal expression gene 3 (Meg3) encodes a non-coding RNA (ncRNA) that plays an important role in the development and progression of cancer. However, the role of Meg3 in glioma cells remains largely unclear. METHODS: Reverse transcription-quantitative (RT-q) PCR was conducted to evaluate the mRNA expression related to cell autophagy and EMT while protein expression was detected by Western blotting. Staining of acidic vacuoles and immunofluorescence staining were used to detect autophagy. The ability of cells to migrate and invade was detected by Transwell migration and invasion assays. RESULTS: In the present study, it was found that the overexpression of Meg3 induced EMT, migration and invasion of glioma cells, whereas Meg3 overexpression induced autophagy of glioma cells. More importantly, the inhibition of autophagy impaired the EMT of glioma cells. In addition, Meg3-induced EMT, migration and invasion could be partially reversed by autophagy inhibitors, chloroquine (CQ) and Lys05, in glioma cells. CONCLUSION: All data suggest that Meg3 induces EMT and invasion of glioma cells via autophagy. Overall, the findings of the present study demonstrate the importance of Meg3 in the molecular etiology of glioma, which also indicate its potential applications in the treatment of glioma.

DNA-templated quantum dots and their applications in biosensors, bioimaging, and therapy.

Over the past 10 years, DNA functionalized quantum dots (QDs) have attracted considerable attention in sensing and imaging of disease-relevant biological targets, as well as cancer therapy. Considerable efforts have been devoted to obtaining DNA functionalized QDs with enhanced stability and quantum yield. Here, we focus on a one-pot method, in which phosphorothioate-modified DNA is used as the co-ligand on the basis of the strong binding of sulfur and Cd2+. After a short summary of the preparation of DNA-templated QDs, versatile bioapplications based on the constructed ratiometric fluorescent probes, nanobeacons and multiple bottom-up assemblies will be discussed. A substantial part of the review will focus on these applications, ranging from small molecule, biological macromolecule, cancer cell and pathogen sensing to in vitro and in vivo imaging. Besides, drug or siRNA delivery based on DNA-templated QD assemblies will also be briefly discussed here.

IFITM3/STAT3 axis promotes glioma cells invasion and is modulated by TGF-ß.

Glioma is the most aggressive primary brain tumor. We have previously provided evidence that IFITM3 promoted glioma cells migration. However, the mechanism of how IFITM3 regulates glioma cells invasion and whether IFITM3 participates in TGF-ß-mediated glioma invasion are still unknown. In this paper, we proved that IFITM3 was notably up-regulated in glioma tissues. Knockdown of IFITM3 suppressed STAT3 phosphorylation in vitro, and a specific STAT3 inhibitor AG490 reversed IFITM3-induced invasion of glioma cells. Furthermore, IFITM3 expression was induced by TGF-ß in glioma and IFITM3 knockdown abolished TGF-ß-mediated glioma cells invasion. Collectively, the results indicate that IFITM3/STAT3 axis may promote TGF-ß-induced glioma cells invasion. This study provided some suggestions for the clinical treatment of the brain tumor.

Organic-inorganic nanoflowers: from design strategy to biomedical applications.

Organic-inorganic hybrid nanoflowers (NF) with sizes or features on a nanoscale are a class of flower-shaped nanomaterials self-assembled from metal ions and organic components. Here, to be more specific, the organic components mainly refer to biomolecules ranging from proteins, peptides, and amino acids to DNA/RNA. Beyond their pleasing aesthetics, their unique properties and integrated functions have attracted widespread interest and made them promising candidates in the application of biomedical areas. Great efforts have been made to design and synthesize versatile functional hybrid nanoflowers. In this review, we begin with the clarification of versatile recently reported hybrid nanoflowers according to the types of metal ions and biomolecules employed. To highlight the design of organic-inorganic hybrid nanoflowers, their synthetic methods and mechanisms, structural and biological characteristics are discussed. After that, the state-of-the-art applications of hybrid nanoflowers in biomedical fields including biosensing, biocatalysis, and cancer therapy are demonstrated. In the end, we discuss the prospects of organic-inorganic hybrid nanoflowers and highlight the challenges and opportunities for future research.

A central role for MeCP2 in the epigenetic repression of miR-200c during epithelial-to-mesenchymal transition of glioma.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) has been linked to the regulation of glioma progression. However, the underlying signaling mechanisms that regulate EMT are poorly understood. METHODS: Quantitative real-time PCR (RT-qPCR) and western blot were performed to detect the expression of MeCP2 in glioma tissues and cell lines. MeCP2 functions were tested with cell immunofluorescence staining and western blot. For in vivo experiments, mouse xenograft model was used to investigate the effects of MeCP2 on glioma. ChIP and Co-IP were used to detect the relationships among MeCP2, miR-200c and Suv39H1. RESULTS: In this study, we found that MeCP2 was frequently up-regulated in human glioma tissues and cell lines. MeCP2 knockdown remarkably induced cell epithelial phenotype and inhibited mesenchymal marker ZEB1 and ZEB2 in vitro and in vivo. In addition, MeCP2 in glioma tissues was negatively correlated with miR-200c expression, and miR-200c overexpression partially abrogated mesenchymal phenotype induced by MeCP2. More importantly, we showed that MeCP2 recruited H3K9 to the promoter of miR-200c by interacting with SUV39H1, resulting in EMT of glioma cells. CONCLUSIONS: This study for the first time reveals MeCP2 as a novel regulator of EMT in glioma and suggest that MeCP2 inhibition may represent a promising therapeutic option for suppressing EMT in glioma.

LncRNA-ATB promotes TGF-ß-induced glioma cells invasion through NF-κB and P38/MAPK pathway.

Glioma constitutes the most aggressive primary intracranial malignancy in adults. We previously showed that long noncoding RNA activated by TGF-ß (lncRNA-ATB) promoted the glioma cells invasion. However, whether lncRNA-ATB is involved in TGF-ß-mediated invasion of glioma cells remains unknown. In this study, quantitative real-time polymerase chain reaction and western blot analysis were used for detecting the mRNA and protein expression of related genes, respectively. Transwell assay was performed to assess the impact of lncRNA-ATB on TGF-ß-induced glioma cells migration and invasion. Immunofluorescence staining was utilized to characterize related protein distribution. Results showed that TGF-ß upregulated lncRNA-ATB expression in glioma LN-18 and U251 cells. Overexpression of lncRNA-ATB activated nuclear factor-κB (NF-κB) pathway and promoted P65 translocation into the nucleus, thus facilitated glioma cells invasion stimulated by TGF-ß. Similarly, lncRNA-ATB markedly enhanced TGF-ß-mediated invasion of glioma cells through activation P38 mitogen-activated protein kinase (P38/MAPK) pathway. Moreover, both the NF-κB selected inhibitor pyrrolidinedithiocarbamate ammonium and P38/MAPK specific inhibitor SB203580 partly reversed lncRNA-ATB induced glioma cells invasion mediated by TGF-ß. Collectively, this study revealed that lncRNA-ATB promotes TGF-ß-induced glioma cell invasion through NF-κB and P38/MAPK pathway and established a detailed framework for understanding the way how lncRNA-ATB performs its function in TGF-ß-mediated glioma invasion.

A fluorometric turn-on aptasensor for mucin 1 based on signal amplification via a hybridization chain reaction and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots.

A fluorometric method is described for the determination of the tumor biomarker mucin 1 (MUC1). It is based on signal amplification of the hybridization chain reaction (HCR), and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots (QDs). If MUC1 bind to the biotin-labeled aptamer, it will initiate HCR with hairpins H1 and H2 to form a long-range dsDNA. The long nucleic acid chains are then linked on the surface of streptavidin-modified magnetic microparticles (MMPs) through streptavidin-biotin interaction. The luminescent ruthenium(II) complex is then embedded in the long dsDNA linked to the MMPs. Hence, there is little Ru complex in the supernatant after magnetic separation, and the fluorescence of the CdZnTeS QDs (best measured at excitation/emission wavelengths of 350/530 nm) is only slightly quenched. In the absence of target, the fluorescence of the CdZnTeS QDs is strongly quenched. Fluorescence increases linearly in the 0.2-100 ng·mL-1 MUC1 concentration range, and the LOD is 0.13 ng·mL-1 (at S/N = 3). The method was applied to the determination of MUC1 in spiked human serum samples. Graphical abstract A fluorometric turn-on aptasensor for mucin 1 is described that is based on the interaction between a Ru(II) complex and quantum dots (QDs). The detection system includes biotin-labeled aptamer-H0, hairpins H1 and H2, streptavidin-modified magnetic microparticles (MMPs), Ru(bpy)2(dppx)2+ and CdZnTeS QDs.

Silicon nanodot-based aptasensor for fluorescence turn-on detection of mucin 1 and targeted cancer cell imaging.

We report herein a new dual-color fluorescent aptasensor for detection of tumor marker mucin 1 (MUC1) and targeted imaging of MCF-7 cancer cells based on the specific interaction between MUC1 and its aptamer S2.2. The aptasensor was prepared by covalent attachment of the cyanine (Cy5)-tagged aptamer S2.2 to fluorescent silicon nanodot (SiND). The fluorescence of S2.2-Cy5 could be quenched by the SiND carrier in the absence of MUC1, and its fluorescence was restored in the presence of MUC1 due to structure switching of S2.2. This aptasensor exhibits specificity for MUC1-possitive MCF-7 cells rather than MUC1-negative MCF-10A cells and Vero cells. The SiND plays multiple roles in this fluorescence assay, making the method easier compared with other approaches. The limit of detection and precision of this method for MUC1 was 1.52 nM and 3.6% (10 nM, n = 7), respectively. The linear range was 3.33-250 nM, and the recoveries in spiked human serum were in the range of 87-108%. This is a simple, selective, sensitive and reliable method, which can well achieve not only quantitative analysis of tumor marker but also dual-color visualization of single cancer cells.

Rational construction of a DNA nanomachine for HIV nucleic acid ultrasensitive sensing.

HIV nucleic acids, one kind of significant biomarker, play an important role in fundamental studies and clinical diagnosis. Importantly, the early accurate diagnosis for HIV nucleic acids at ultralow concentrations can potentially extend the life of patients. In the current work, we developed a DNA nanomachine on gold nanoparticles (AuNPs) coupling rolling circle amplification and DNA walker cascade amplification for ultrasensitive detection of HIV nucleic acids. This DNA nanomachine sensing strategy exhibits a significantly low detection limit down to 1.46 fM. Furthermore, this DNA nanomachine biosensor is capable of detecting target DNA in real samples because of its high selectivity and sensitivity. Moreover, the DNA nanomachine biosensor is capable of discriminating single-base mismatch lower than 3.5 pM. The results showed that this DNA nanomachine biosensor has the potential for biomedical studies and clinical applications.

Sensitive fluorescent detection of methyltransferase based on thermosensitive poly(N-isopropylacrylamide).

DNA methyltransferase (MTase) has a crucial role in many biological processes, its abnormal expression level has been regarded as a predictive cancer biomarker. Herein, a sensitive fluorescence method based on thermosensitive poly (N-isopr-opylacrylamide) was developed to assay of M.SssI activity. When the M.SssI was introduced, dsDNA was methylated at palindromic sequence 5'-CmCGG-3' and became resistant to cleavage by the endonuclease HpaII. Therefore, a biotin modified ssDNA and a FAM modified ssDNA were designed including the recognized sites for both methyltransferase M.SssI and endonuclease HpaII. By SA-biotin intereaction, the DNA was conjugated to thermosensitive poly (N-isopropylacrylamide) modified by SA, the methylated substrate fluorescence was increased with the concentration of M.SssI increasing. The proposed method has a low detection limit of 0.18 U/mL. This simple method can be a useful tool to apply in diagnosis and biomedical research, which was successfully investigated in the serum sample.

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis.

Label-free silicon nanodots featured ratiometric fluorescent aptasensor for lysosomal imaging and pH measurement.

The homeostasis of lysosomal pH is crucial in cell physiology. Developing small fluorescent nanosensors for lysosome imaging and ratiometric measurement of pH is highly demanded yet challenging. Herein, a pH-sensitive fluorescein tagged aptamer AS1411 has been utilized to covalently modify the label-free fluorescent silicon nanodots via a crosslinker for construction of a ratiometric pH biosensor. The established aptasensor exhibits the advantages of ultrasmall size, hypotoxicity, excellent pH reversibility and good photostability, which favors its application in an intracellular environment. Using human breast MCF-7 cancer cells and MCF-10A normal cells as the model, this aptasensor shows cell specificity for cancer cells and displays a wide pH response range of 4.5-8.0 in living cells. The results demonstrate that the pH of MCF-7 cells is 5.1, which is the expected value for acidic organelles. Lysosome imaging and accurate measurement of pH in MCF-7 cells have been successfully conducted based on this nanosensor via fluorescent microscopy and flow cytometry.

Autofluorescent gelatin nanoparticles as imaging probes to monitor matrix metalloproteinase metabolism of cancer cells.

In this paper, autofluorescent gelatin nanoparticles were synthesized as matrix metalloproteinase (MMP) responsive probes for cancer cell imaging. A modified two-step desolvation method was employed to generate these nanoparticles whose size was controllable and had stable autofluorescence. As glutaraldehyde was introduced as the crosslinking agent, the generation of Schiff base (CN) and double carbon bond (CC) between glutaraldehyde and gelatin endowed these gelatin nanoparticles distinct autofluorescence. Considering MMPs were usually overexpressed on the surface of cancer cells and they had degradation ability toward gelatin, we utilized these nanoparticles as imaging probes to responsively monitor the MMP metabolism of cancer cells according to the luminance change. As fluorescent probes, these nanoparticles had facile synthesis procedure and good biocompatibility, and provided a smart strategy to monitor cancer cell behaviors. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2854-2860, 2016.

Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin.

In this work, a versatile dumbbell molecular (DM) probe was designed and employed in the sensitively homogeneous bioassay. In the presence of target molecule, the DM probe was protected from the digestion of exonucleases. Subsequently, the protected DM probe specifically bound to the intercalation dye and resulted in obvious fluorescence signal which was used to determine the target molecule in return. This design allows specific and versatile detection of diverse targets with easy operation and no sophisticated fluorescence labeling. Integrating the idea of target-protecting DM probe with adenosine triphosphate (ATP) involved ligation reaction, the DM probe with 5'-end phosphorylation was successfully constructed for ATP detection, and the limitation of detection was found to be 4.8 pM. Thanks to its excellent selectivity and sensitivity, this sensing strategy was used to detect ATP spiked in human serum as well as cellular ATP. Moreover, the proposed strategy was also applied in the visual detection of ATP in droplet-based microfluidic platform with satisfactory results. Similarly, combining the principle of target-protecting DM probe with streptavidin (SA)-biotin interaction, the DM probe with 3'-end biotinylation was developed for selective and sensitive SA determination, which demonstrated the robustness and versatility of this design.

Multifunctional Dumbbell-Shaped DNA-Templated Selective Formation of Fluorescent Silver Nanoclusters or Copper Nanoparticles for Sensitive Detection of Biomolecules.

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine-thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation. By carefully investigating the sequence and length of DS DNA, we present the optimal design of the template. Benefiting from the smart design and facile synthesis, a simple, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection is proposed. Through the systematic comparison, it is found that the strategy based on CuNPs formation is more sensitive for ATP assay than that based on AgNCs synthesis, and the detection limitation was found to be 81 pM. What's more, the CuNPs formation-based method is successfully applied in the detection of ATP in human serum as well as the determination of cellular ATP. In addition to small target molecule, the sensing strategy was also extended to the detection of biomacromolecule (DNA), which illustrates the generality of this biosensor.

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins.

An amplified colorimetric method has been developed for the detection of protein and cancer cells based on the assembly of nucleic acids and proteins for the first time. In this process, the assembly of nucleic acids was triggered by a biotinylated DNA strand after a sandwich immunoreaction. The biotinylated DNA strand and sandwich immunocomplex were connected by streptavidin. Then, the assembly of biotinylated bovine serum albumin (Biotin-BSA) and streptavidin-horseradish peroxidase (SA-HRP) occurred at a node of the assembled products of nucleic acids through the biotin-streptavidin reaction. Under the catalysis of horseradish peroxidase, 3,3',5,5'-tetramethylbenzidine (TMB) was oxidized by H2O2 and the oxidized product was analyzed by its UV-vis absorbance signal and sensitive colorimetric detection. This colorimetric sensor could not only achieve the quantitative determination of protein by UV-vis absorbance but could also be applied for semiquantitative determination by digital visualization. Using alpha-fetoprotein (AFP) as the model target, this proposed colorimetric method showed a wide linear range from 5 pg/mL to 1 ng/mL with a detection limit of 1.95 pg/mL by the instrument, and even 5 pg/mL target protein could be distinguished simply by the naked eye. This approach was then expanded to detect cancer cells based on the recognition of folic acid receptors that were over-expressed on the cancer cells by folic acid-tethered DNA. More importantly, this strategy can be further used as a universal colorimetric method for the determination of viruses or other proteins by changing the corresponding antibodies.

Rolling cycle amplification based single-color quantum dots-ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA.

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots-ruthenium complex (QDs-Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)2(dppx)](2+) (dppx=7,8-dimethyldipyrido [3,2-a:2',3'-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)2(dppx)](2+) is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)2(dppx)](2+) through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs-Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.

A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I.

We have developed a new fluorescence method for specific single-stranded DNA sequences with exonuclease III (Exo III) and nucleic acid dye SYBR Green I. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 27 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of the target DNA, the hairpin-probe is in the stem-closed structure, the fluorescence of SYBR Green I is very strong. In the presence of the target DNA, the hairpin-probe hybridizes with the target DNA to form double-stranded structure with a blunt 3'-terminus. Thus, in the presence of Exo III, only the 3'-terminus of probe is subjected to digestion. Exo III catalyzes the stepwise removal of mononucleotides from this terminus, releasing the target DNA. The released target DNA then hybridizes with another probe, whence the cycle starts anew. The signal of SYBR Green I decreases greatly. This system provides a detection limit of 160 pM, which is comparable to the existing signal amplification methods that utilized Exo III as a signal amplification nuclease. Due to the unique property of Exo III, this method shows excellent detection selectivity for single-base discrimination. More importantly, superiors to other methods based on Exo III, these probes have the advantages of easier to design, synthesize, purify and thus are much cheaper and more applicable. This new approach could be widely applied to sensitive and selective nucleic acids detection.

An ultrasensitive biosensor for DNA detection based on hybridization chain reaction coupled with the efficient quenching of a ruthenium complex to CdTe quantum dots.

A highly sensitive and selective DNA biosensor based on hybridization chain reaction is described, which combines CdTe quantum dots (QDs) and a ruthenium complex. Based on the variation of fluorescence signals of the CdTe QDs, the target DNA is determined.