DNA nanostructures for exploring cell-cell communication.

The process of coordinating between the same or multiple types of cells to jointly execute various instructions in a controlled and carefully regulated environment is a very appealing field. In order to provide clearer insight into the role of cell-cell interactions and the cellular communication of this process in their local communities, several interdisciplinary approaches have been employed to enhance the core understanding of this phenomenon. DNA nanostructures have emerged in recent years as one of the most promising tools in exploring cell-cell communication and interactions due to their programmability and addressability. Herein, this review is dedicated to offering a new perspective on using DNA nanostructures to explore the progress of cell-cell communication. After briefly outlining the anchoring strategy of DNA nanostructures on cell membranes and the subsequent dynamic regulation of DNA nanostructures, this paper highlights the significant contribution of DNA nanostructures in monitoring cell-cell communication and regulating its interactions. Finally, we provide a quick overview of the current challenges and potential directions for the application of DNA nanostructures in cellular communication and interactions.

Spatial Control of Receptor Dimerization Using Programmable DNA Nanobridge.

Receptor dimerization is an essential mechanism for the activation of most receptor tyrosine kinases by ligands. Thus, regulating the nanoscale spatial distribution of cell surface receptors is significant for studying both intracellular signaling pathways and cellular behavior. However, there are currently very limited methods for exploring the effects of modulating the spatial distribution of receptors on their function by using simple tools. Herein, we developed an aptamer-based double-stranded DNA bridge acting as "DNA nanobridge", which regulates receptor dimerization by changing the number of bases. On this basis, we confirmed that the different nanoscale arrangements of the receptor can influence receptor function and its downstream signals. Among them, the effect gradually changed from helping to activate to inhibiting as the length of DNA nanobridge increased. Hence, it can not only effectively inhibit receptor function and thus affect cellular behavior but also serve as a fine-tuning tool to get the desired signal activity. Our strategy is promising to provide insight into the action of receptors in cell biology from the perspective of spatial distribution.

Magnetic-Nanowaxberry-Based Simultaneous Detection of Exosome and Exosomal Proteins for the Intelligent Diagnosis of Cancer.

Exosome concentration and exosomal proteins are regarded as promising cancer biomarkers. Herein, a waxberry-like magnetic bead (magnetic-nanowaxberry) which has huge surface area and strong affinity was synthesized to couple with aptamer for exosome capture and recovery. Subsequently, we developed a fluorescent assay for the sensitive, accurate, and simultaneous quantification of exosome and cancer-related exosomal proteins [epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM)] by using triple-colored probes to recognize EGFR and EpCAM or spontaneously anchor to the lipid bilayer. In this design, the interference of soluble proteins can be avoided due to the dual recognition strategy. Moreover, the lipid-based quantification of exosome concentration can improve the accuracy. Besides, the simultaneous detection mode can save samples and simplify the operation steps. Consequently, the assay shows high sensitivity (the limits of detection are down to 0.96 pg/mL for EGFR, 0.19 pg/mL for EpCAM, and 2.4 × 104 particles/µL for exosome), high specificity, and satisfactory accuracy. More importantly, this technique is successfully used to analyze exosomes in plasma to distinguish cancer patients from healthy individuals. To improve the diagnostic efficacy, the deep learning was used to exploit the potential pattern hidden in data obtained by the proposed method. Also, the accuracy for the intelligent diagnosis of cancer can achieve 96.0%. This study provides a new avenue for developing new biosensors for exosome analysis and intelligent disease diagnosis.

In situ detection of plasma exosomal microRNA for lung cancer diagnosis using duplex-specific nuclease and MoS2 nanosheets.

MicroRNAs (miRNAs) encapsulated in tumor-derived exosomes are becoming ideal biomarkers for the early diagnosis and prognosis of lung cancer. However, the accuracy and sensitivity are often hampered by the extraction process of exosomal miRNA using traditional methods. Herein, this study developed a fluorogenic quantitative detection method for exosomal miRNA using the fluorescence quenching properties of molybdenum disulfide (MoS2) nanosheets and the enzyme-assisted signal amplification properties of duplex-specific nuclease (DSN). First, a fluorescently-labeled nucleic acid probe was used to hybridize the target miRNA to form a DNA/RNA hybrid structure. Under the action of the DSN, the DNA single strand in the DNA/RNA hybrid strand was selectively digested into smaller oligonucleotide fragments. At the same time, the released miRNA target triggers the next reaction cycle, so as to achieve signal amplification. Then, MoS2 was used to selectively quench the fluorescence of the undigested probe leaving the fluorescent signal of the fluorescently-labeled probe fragments. The fluorometric signals for miRNA-21 had a maximum excitation/emission wavelength of 488/518 nm. Most importantly, the biosensor was then applied for the accurate quantitative detection of miRNA-21 in exosome lysates extracted from human plasma and this method was able to successfully distinguish lung cancer patients from healthy people. This biosensor provides a simple, rapid, and a highly specific quantitative method for exosomal miRNA and has promising potential to be used in the early diagnosis of lung cancer.

Fluorometric immunoassay for the simultaneous determination of the tumor markers carcinoembryonic antigen and cytokeratin 19 fragment using two kinds of CdSe/ZnS quantum dot nanobeads and magnetic beads.

A method is described for the simultaneous determination of the carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1). Two kinds of CdSe/ZnS quantum dot nanobeads (QBs), with emission maxima at 530 nm (green) and 585 nm (yellow), were used as labels, and magnetic beads (MBs) for separation. The MBs were used as substrates to couple CEA and CYFRA21-1 antibody for isolating the proteins. Then, the differently colored QBs were linked to the antibodies against CEA and CYFRA21-1, respectively. Following the formation of the immunocomplex, the intensities of the green and yellow emissions were measured at the same excitation wavelength of 340 nm. The detection limits are 0.1 ng⋅mL- 1 for CEA, and of 0.2 ng⋅mL- 1 for CYFRA21-1. The recoveries from spiked serum are 92.1 - 118.1% for CEA, and from 90.8% to 115.2% for CYFRA21-1, with the relative standard deviations of 6.3 - 12.3% and 7.1 - 11.8%. The method was successfully applied to the simultaneous determination of the two proteins in human serum sample (n = 45). The results correlated well with those of the chemiluminescent enzyme immunoassay kit. Graphical AbstractSchematic presentation of the fluorescence immunoassay for the simultaneous determination of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1) based on quantum dot nanobeads (QBs) and magnetic beads (MBs) is reported. The intensities of two kinds of CdSe/ZnS QBs, with the emission maxima at 530 nm (green) and 585 nm (yellow) were measured at the same excitation wavelength of 340 nm.

The structural characteristics of mononuclear-macrophage membrane observed by atomic force microscopy.

Mononuclear macrophages are important immune cells in the organisms. The complicated membrane structure underlying the diverse functions of mononuclear-macrophage has been largely unresolved. As a representative of monocyte-derived macrophages, the membrane structure of PMA differentiated THP-1 cells was comprehensively investigated by AFM-based single molecule approaches. The rugged ectoplasmic side of mononuclear-macrophage membrane are significantly different from erythrocytes and mammalian somatic cell membranes. But the smooth lipid bilayer and the branched lipid raft domains obtained by proteinase K and MßCD treatment of the protein-covered cytoplasmic side, are common characteristics among all the studied cell membranes. This discovery of distinct organization of membrane proteins on both sides of mononuclear-macrophage membranes provides additional evidence for the asymmetry of membrane structure. The podosome-associated structures of mononuclear-macrophage were directly examined, and the independent localization of podosome domains and the lipid rafts was verified by in situ AFM, giving new insight into this multifunctional organelle.

Simultaneous detection of carcinoembryonic antigen and neuron-specific enolase in human serum based on time-resolved chemiluminescence immunoassay.

In the clinical diagnosis of tumor, the immunological detection of single tumor markers may lead to errors and missed inspection. Therefore, it is necessary to establish an accurate and effective method for the simultaneous detection of multiple tumor markers. Thus, we developed a time-resolved chemiluminescence immunoassay (TRCLIA) to simultaneously detect carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) in human serum. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were used as the detection probes to label the monoclonal antibodies of CEA and NSE by strain-promoted azide-alkyne cycloaddition (SPAAC), respectively. Based on a sandwich immunoassay, the targets in the samples were captured by antibodies immobilized on the surface of carboxylate-modified polystyrene microspheres (CPSMS) and sandwiched by other antibodies labeled with HRP and ALP. Since HRP and ALP had different dynamic characteristics, the CEA and NSE signals were recorded at 0.5 s and 20 min, respectively, and cross-interference could be avoided effectively. The whole signal detection processes could be completed in 20 min. The linear ranges of CEA and NSE were 0.1-64 ng mL-1 and 0.05-64 ng mL-1 and the limits of detection were 0.085 ng mL-1 and 0.044 ng mL-1 (S/N = 2), respectively. Also, 45 human serum samples obtained from patients having lung disease were tested by TRCLIA and commercial chemiluminescence enzyme-linked immunoassay (CLEIA) kits with good correlation. The correlation coefficients of CEA and NSE were 0.985 and 0.970, respectively. The results demonstrated a novel, effective, reliable and convenient TRCLIA method for the clinical diagnosis of CEA and NSE. The TRCLIA method has the potential to be an effective clinical tool for the early screening of lung cancer and can be applied in clinical diagnosis.

Enhanced chemiluminescence enzyme-linked immunoassay for the determination of DNA methyltransferase 1 in human serum.

The occurrence of many diseases is closely related to the high expression of DNA methyltransferase 1 (DNMT1). However, most studies are focused on the detection of DNMT1 activity, a few are concerned with the detection of DNMT1 content. In this study, we developed a simple and highly sensitive chemiluminescence (CL) assay for the detection of DNMT1 content. In this method, anti-DNMT1 monoclonal antibody was coated on a polystyrene microplate to capture DNMT1. Then anti-DNMT1 polyclonal antibody and goat anti-rabbit immunoglobulin G with horseradish peroxidase (IgG-HRP) were respectively added to combine with captured DNMT1 to form a sandwich structure. Finally, the HRP could catalyze CL substrate and achieve CL signal response. Based on this novel sensitive strategy, the recovery percents were in the ranges from 71.5% to 91.0%. The precision of intra-assays and inter-assays were 5.45%-11.29% and 7.03%-11.25%, respectively. The method was successfully applied for the determination of DNMT1 in human serum. The detection results of serum samples showed that the proposed assay had a high correlation with enzyme-linked immunosorbent assay (ELISA) kit. Compared with the ELISA kit (limit of detection = 0.1 ng/mL), the method has a lower limit of detection of 0.042 ng/mL. Therefore, our method has the potential for the detection of DNMT1 content in clinical diagnosis.

An ultrasensitive chemiluminescence immunoassay for fumonisin B1 detection in cereals based on gold-coated magnetic nanoparticles.

BACKGROUND: In the present study, a novel highly sensitive magnetic enzyme chemiluminescence immunoassay (MECLIA) was developed to detect fumonisin B1 (FB1 ) in cereal samples. The gold-coated magnetic nanoparticles (Fe3 O4 @Au, GoldMag) were used as solid phase carrier to develop a competitive CLIA for detecting FB1 , in which FB1 in samples would compete with FB1 -ovalbumin coated on the surface of Fe3 O4 @Au nanoparticles for binding with FB1 antibodies. Successively, horseradish peroxidase labeled goat anti-rabbit IgG (HRP-IgG) was conjugated with FB1 antibodies on the microplate. In substrate solution containing luminol and H2 O2 , HRP-IgG catalyzed luminol oxidation by H2 O2 , generating a high chemiluminescence signal. The FB1 immune GoldMag particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscope and zeta potential analysis, etc. RESULTS: The concentrations and the reaction times of these immunoreagents were optimized to improve the performances of this method. The established method could detect as low as 0.027 ng mL-1 FB1 from 0.05 ng mL-1 to 25 ng mL-1 , demonstrating little cross-reaction (less than 2.4%) with other structurally related compounds. The average intrassay relative SD (RSD) (n = 6) was 3.4% and the average interassay RSD (n = 6) was 5.4%. This method was successfully applied for the determination of FB1 in corn and wheat and gave recoveries of between 98-110% and 91-105%, respectively. CONCLUSION: The results of the present study suggest that the MECLIA approach has potential application for high-throughput fumonisin screening in cereals. © 2018 Society of Chemical Industry.

Competitive host-guest interaction between ß-cyclodextrin polymer and pyrene-labeled probes for fluorescence analyses.

We developed a novel homogeneous fluorescence analysis based on a novel competitive host-guest interaction (CHGI) mechanism between ß-cyclodextrin polymer (polyß CD) and pyrene-labeled probe for biochemical assay. Pyrene labeling with oligonucleotide strands can be recruited and reside in lipophilic cavities of polyß CD. This altered lipophilic microenvironment provides favored polarity for enhanced quantum efficiencies and extraordinarily increases the luminescence intensity of pyrene. However, with addition of complementary DNA, the pyrene-labeled probe formed double-strand DNA to hinder pyrene from entering the cavities of polyß CD. The release of pyrene from polyß CD, which are followed by fluorescence extinguishing, will provide the clear signal turn-off in the presence of target DNA. We also introduced Exodeoxyribonuclease I (Exo I) and Exodeoxyribonuclease III (Exo III) to improve the sensitivity of this system, and the following product of cleavage reaction, pyrene-nucleotide, could more easily host-guest interact with polyß CD and emit stronger fluorescence than pyrene-labeled probe. In addition, the successful detection of adenosine is also demonstrated by using the similar sensing scheme. Although this scheme might be easily interfered by some biomolecules in the real test sample, it holds promising potential for detecting a broad range of other types of aptamer-binding chemicals and biomolecules.

Self-assembled supramolecular nanoprobes for ratiometric fluorescence measurement of intracellular pH values.

Self-assembly of small building blocks into functional supramolecular nanostructure has opened prospects for the design of novel materials. With this molecular engineering strategy, we have developed self-assembled supramolecular nanoprobes (SSNPs) for ratiometric fluorescence measurement of pH values in cells. The nanoprobes with a diameter of ∼30 nm could be formulated just by mixing pH-sensitive adamantane-fluorescein (Ad-F) and pH-insensitive adamantane-Rhodamine B (Ad-R) with ß-cyclodextrin polymer (poly-ß-CD) at one time. The nanoprobes with good biocompatibility have been successfully applied to measure intracellular pH in the pH range of 4-8 and estimate pH fluctuations associated with different stimuli in cells. Moreover, we expect that this self-assembled approach is applicable to the construction of nanoprobes for other targets in cells just by replacing the respective indicator dyes with relevant indicators.

Poly ß-cyclodextrin/TPdye nanomicelle-based two-photon nanoprobe for caspase-3 activation imaging in live cells and tissues.

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. ß-cyclodextrin polymer (ßCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/ßCDP nanomicelle with the TPE technique, herein we have designed a TPdye/ßCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/ßCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/ßCDP nanomicelle's surface to form TPA DEASPI/ßCDP@Ad-DEVD-BHQ2 nanoconjugate by the ßCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/ßCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/ßCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/ßCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

A novel ß-cyclodextrin-assisted enhancement strategy for portable and sensitive detection of miR-21 in human serum.

Benefiting from our discovery that ß-cyclodextrin (ß-CD) could enhance the catalytic activity of invertase through hydrogen bonding to improve detection sensitivity, a highly sensitive and convenient biosensor for the detection of miR-21 was proposed, which is based on the simplicity of reading signals from a personal glucose meter (PGM), combined with self-assembled signal amplification probes and the performance of ß-CD as an enhancer. In the presence of miR-21, magnetic nanoparticle coupled capture DNA (MNPs-cDNA) could capture it and then connect assist DNA/H1-invertase (aDNA/H1) and self-assembled signal amplification probes (H1/H2) in turn. As a result, a "super sandwich" structure was formed. The invertase on MNPs-cDNA could catalyze the hydrolysis of sucrose to glucose and this catalytic process could be enhanced by ß-CD. The PGM signal exhibited a linear correlation with miR-21 concentration within the range of 25 pmol L-1 to 3 nmol L-1, and the detection limit was as low as 5 pmol L-1 with high specificity. Moreover, the recoveries were 103.82-124.65% and RSD was 2.59-6.43%. Furthermore, the biosensor was validated for the detection of miR-21 in serum, and the results showed that miR-21 levels in serum samples from patients with Diffuse Large B-Cell Lymphoma (DLBCL) (n = 12) were significantly higher than those from healthy controls (n = 12) (P < 0.001). Therefore, the ingenious combination of PGM-based signal reading, self-assembled signal amplification probes and ß-CD as an enhancer successfully constructed a convenient, sensitive and specific biosensing method, which is expected to be applied to clinical diagnosis.

Enzyme-free colorimetric detection of DNA by using gold nanoparticles and hybridization chain reaction amplification.

A novel, high sensitive, and specific DNA assay based on gold nanoparticle (AuNP) colorimetric detection and hybridization chain reaction (HCR) amplification has been demonstrated in this article. Two hairpin auxiliary probes were designed with single-stranded DNA (ssDNA) sticky ends which stabilize AuNPs and effectively prevent them from salt-induced aggregation. The target DNA hybridized with the hairpin auxiliary probes and triggered the formation of extended double-stranded DNA (dsDNA) polymers through HCR. As a result, the formed dsDNA polymers provide less stabilization without ssDNA sticky ends, and AuNPs undergo aggregation when salt concentration is increased. Subsequently, a pale purple-to-blue color variation is observed in the colloid solution. The system is simple in design and convenient in operation. The novel strategy eliminates the need for enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentation. The detection and discrimination process can be seen with the naked eye. The detection limit of this method is lower than or at least comparable to previous AuNP-based methods. Importantly, the protocol offers high selectivity for the determination between perfectly matched target oligonucleotides and targets with single base-pair mismatches.

Biomimetic-compartmented nanoprobe for in-situ imaging of iron storage and release from ferritin in cells.

Ferritin plays an important role in regulating the homeostasis of iron in cells by storing/releasing iron. Current methods usually explored the determination of iron content, but in-situ imaging of the iron storage/release from ferritin in cells cannot be achieved. Hence, an engineered self-assembled biomimetic-compartmented nanoprobe (APO@CDs) has been constructed. The protein shell of APO (apoferritin) acted as ion channel module to control iron ions entering/exiting ferritin cavity; the inner core of CDs (carbon dots) acted as signal module for iron ions response. Compared with CDs, the response sensitivity and specificity to iron ions (Fe3+) have been improved by using APO@CDs, and the cytotoxicity was significantly reduced. Additionally, compared with cells containing APO@CDs alone, the normalized fluorescence gray value of Fe3+-treated cells was significantly decreased (0.275), indicating that Fe3+ has effectively entered the ferritin. Furtherly, that of Fe3+-treated cells incubated with deferoxamine (DFO) was significantly enhanced (0.712), showing that Fe3+ was released from ferritin under the mediation of DFO. The results demonstrate that APO@CDs can be successfully applied to in-situ imaging of iron storage/release from ferritin in cells, providing a potential platform for the in-situ dynamic study of the iron storage/release in biomedical field.

Activatable Fluorescence-Encoded Nanoprobes Enable Simple Multiplexed RNA Imaging in Live Cells.

Benefiting from superior programmable performance and flexible design of DNA technologies, a variety of single-molecule RNA fluorescence imaging methodologies have been reported. However, the multiplexing capability is restricted owing to the spectral overlap of fluorophores. To overcome this limitation, some inspiring multiplex imaging strategies have been developed, but in practice, it remains challenging to achieve convenient and rapid imaging in live cells due to complex designs and additional pretreatments to increase cell permeability. Here, we report an activatable fluorescence-encoded nanoprobe (AFENP) strategy, through which fluorescence-encoded functional modules for qualitative analysis and activated nucleic acid assemblies functional modules for quantitative testing enable simple multiplexed RNA imaging in single live cells. As a proof of principle, by two distinguishable fluorophores (fluorescein and rhodamine B) and their seven distinctly differentiated intensity levels, self-assembled AFENP enables simplified and quick simultaneous in situ detection and imaging of seven types of targets in live single cells because the fluorescent quantitative signal is activated only in the presence of target avoiding the washing procedures and additional pretreatment to increase cell permeability is undesired. We expect that this practical single-cell analysis platform will be adopted for multiple gene expression analysis and imaging in live cells on account of its simplicity and multiplex capability.

Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer.

Exosomal proteins are considered to be promising indicators of cancer. Herein, a novel DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a strategy was proposed for the synchronous determination of exosomal proteins: serum amyloid A-1 protein (SAA1) and coagulation factor V (FV). In this design, the paired antibodies were used to recognize targets, thereby ensuring the specificity. DNAzyme walkers were employed to convert the contents of SAA1 and FV into activators (P1 and P2), and one target can produce abundant activators, thus achieving an initial amplification of signal. Furthermore, the P1 and P2 can activate CRISPR-Cas12a/Cas13a system, which in turn trans-cleaves the reporters, enabling a second amplification and generating two fluorescent signals. The assay is highly sensitive (limits of detection as low as 30.00 pg/mL for SAA1 and 200.00 pg/mL for FV), highly specific and ideally accurate. More importantly, it is universal and can be used to detect both non-membrane and membrane proteins in exosome. Besides, the method can be successfully applied to detect SAA1 and FV in plasma exosomes to differentiate between lung cancer patients and healthy individuals. To explore the application of the developed method in tumor diagnosis, a deep learning model based on the expressions of SAA1 and FV was developed. The accuracy of this model can achieve 86.96%, which proves that it has a promising practical application capacity. Thus, this study does not only provide a new tool for the detection of exosomal proteins and cancer diagnosis, but also propose a new strategy to detect non-nucleic acid analytes for CRISPR-Cas system.

Fluorescence quenching determination of tetracyclines based on the synergistic oxidation effect between Fe3O4nanoparticles and tetracyclines.

In recent years, tetracyclines (TCs) is a hot research topic. Herein, we report an interesting discovery using the complexation of oxytetracycline and metal ions. In this study, according to the properties of Fe3O4nanoparticles (Fe3O4NPs) as a nanoenzyme, it can be used to catalyze the oxidation of KI by H2O2to produceI3-,while at the same timeI3-binds to rhodamine 6G (Rh6G) to form a conjoined particle (Rh6G ∼ I3)n, leading to a decrease in the fluorescence intensity of Rh6G. However, in the presence of TCs, Fe3O4NPs have a synergistic effect with TCs, leading to enhanced catalytic activity, as well as better selectivity compared to the activity of other reducing enzymes. Consequently,the fluorescent signal based on a resonance scattering effect between Rh6G andI3-is dependent on the concentration of TCs, thus achieving highly facile and robust detection of TCs. The limits of detection (LOD) of the method were 20 nM, 10 nM and 40 nM for oxytetracycline(OTC), tetracycline(TC) and chlortetracycline(CTC), respectively. Most importantly, the method can be successfully applied to the detection of TCs in milk, eggs, and honey. The recoveries of spiked samples ranged from 83.11 to 118.95%. Thus, a stable, hands-on strategy for the detection of TCs is proposed, which has potential applications in the field of food safety and environmental protection.

A digital immuno-PCR assay for simultaneous determination of 5-methylcytosine and 5-hydroxymethylcytosine in human serum.

This work aimed to develop an ultrasensitive and specific immunosorbent assay for simultaneous detection of double DNA methylation marks. Being considered the most important indicators in disease diagnosis, clinical treatment, and prognosis, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were chosen as the proof-of-concept targets. The described strategy consisted of Phos-tag Biotin anchoring at streptavidin-magnetic nanoparticles, specific immune recognition of anti-5mC antibody and anti-5hmC antibody and labeling of Barcode-antibody, signal amplification of immune PCR and digital PCR machine. Under optimal conditions, the digital immuno-PCR assay showed a board dynamic range from 2.7 × 10-13 mol/L to 2.7 × 10-9 mol/L and the detection limits were 61.7 fmol/L for 5mC, and of 0.111 pmol/L for 5hmC. A 16-fold and 186-fold improvement of LOD were obtained by the proposed approach for 5mC and 5hmC detection compared with real-time immune PCR. The approach also showed ideal specificity, repeatability and stability. The recovery test demonstrated that the digital immuno-PCR assay is a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample.