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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.

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.

Background As two important tumor markers, vascular endothelial growth factor (VEGF) and carcinoembryonic antigen (CEA) have a great value for clinical application in the early diagnosis of cancer. Due to the complex composition of biological samples, the results from combined detection of CEA and VEGF are often taken as a comprehensive indicator in order to make an accurate judgment on a disease. However, most of the current methods can only be used to detect the content of one biomarker. Therefore, it is necessary to explore a simple, rapid, low-cost, and highly sensitive method for the simultaneous detection of CEA and VEGF. Methods Based on specific aptamers and magnetic separation, a time-resolved chemiluminescence enzyme-linked aptamer assay was developed for the simultaneous detections of CEA and VEGF in serum samples. Results Under the optimal conditions, the linear range of the calibration curve for VEGF was from 0.5 to 80 ng mL−1, and the limit of detection was 0.1 ng mL−1. The linear range of the calibration curve for CEA was 0.5 to 160 ng mL−1, and the limit of detection was 0.1 ng mL−1. The established method was applied to detect VEGF and CEA in serum samples. The results were consistent with those of commercial kits. Conclusion The method has high sensitivity and can quickly obtain accurate results, which could greatly improve the measurement efficiency, reduce the cost, and also reduce the volume of sample consumed. It can be seen that the method established in this study has important application value and broad application prospect in clinical diagnosis.

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

DNA methyltransferase 1 (DNMT1) is a useful biomarker for lung cancer in early clinical diagnosis. A rapid magnetic chemiluminescence immunoassay (MCLIA) for DNMT1 in human serum has been developed. Horseradish peroxidase (HRP)-second-Ab was used to labeled polyclonal antibodies of anti-DNMT1. DNMT1 in sample integrates with specific immunomagnetic beads and can constitute a supersandwiched immunoreaction. In magnetic field, nonspecific materials can be separated. After luminescent substrate luminol-H2O2-BIP was added, the relative light unit (RLU) of HRP was detected and was discovered to be directly proportional to the content of DNMT1 in sample. The correlative variables involved in the MCLIA value were optimized and the methodological evaluation was carried out. After optimization, in the range of 0.5–128 ng/mL, the linear regression equation was y = 0.5014x + 1.769 (x was logCDNMT1, y was relative luminescence units (RLU)/RLU0), and the limit of detection was 0.01 ng/mL. The RSD of intra- and inter-assays were 15.8%–16.9% and 14.3%–18.1%, respectively. The recovery was from 70.0% to 106.2%. Furthermore, paralleled with purchasable enzyme-linked immunosorbent assay (ELISA) kits, MCLEIA had lower detection limit, wider linear range and shorter detection time. Therefore, the MCLEIA established in this study could be used for the sensitive detection of DNMT1 in serum sample.

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.

The establishment of a highly sensitive and efficient platform for detecting circulating miRNA in serum is important for disease diagnosis and prognosis. To this end, a self-assembled horseradish peroxidase (HRP)-based polymer (poly-HRP) dual signal amplification strategy, via skillful combination of functionalized magnetic nanoparticles as the capture nanoprobes and the poly-HRP as signal amplification probes, has been developed for highl-sensitive and accurate detection of circulating miR-142-3p in human serum. Just by mixing building blocks together at one time, the poly-HRP used for dual signal amplification can be engineered by only one-step assembly. As the poly-HRP can be directly prepared ahead, avoiding chain amplification reaction in the detection process, the assay can be rapidly accomplished in about one hour. The measured limit-of-detection (LOD) is down to 100 fM. Standard miR-142-3p spiked in human serum is measured with ideal recovery rates ranging from 97.46% to 115.90%. More importantly, this method was validated to be applicable to direct detection of miR-142-3p in serums, and the results suggested that the miR-142-3p levels of serum samples obtained from T-cell acute lymphoblastic leukemia children (n = 25) are significantly higher than those from healthy control groups (n = 25) (P

Acatastatic pH values of organelles are associated with cell dysfunction and various diseases, which may increase the risk of diabetes, obesity, cancer, Alzheimer's disease and so on. Thus, in situ monitoring of the pH of organelles is extremely essential. Herein, we designed ratiometric fluorescent nanoprobes based on self-assembly for imaging of mitochondrial pH in living cells. The nanoprobes consisted of three parts: (1) a β-cyclodextrin polymer (β-CDP) acted as the backbone of the nanoprobes; (2) two lipophilic dyes acted as the pH sensitive unit and reference unit, i.e. adamantane-labeled fluorescein (Ad-F) and adamantane-labeled rhodamine (Ad-R); (3) adamantane-labeled triphenylphosphonium (Ad-TPP) acted as the mitochondrial targeting unit. Confocal microscopy images proved that the nanoprobes have been successfully applied to monitor intracellular pH in the range of pH 4.0–8.0. Moreover, as evidenced by co-localization imaging, the nanoprobes also showed good ability to target mitochondria (Pearson correlation coefficient was 0.88). Additionally, the nanoprobes showed low toxicity, excellent pH reversibility, and flexibility of construction, and have the potential to monitor pH of other organelles if different targeting units are conjugated. Our strategy is expected to provide new tools for pH monitoring in specific organelles and diagnosis of pH related diseases.

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.

Deoxynivalenol (DON), Fumonisin B1 (FB1) and Aflatoxin B1 (AFB1) have strong toxicity to humans and exist widely in grain and food. It is necessary to screen these mycotoxins to guarantee food safety. In order to develop a rapid, simple, low-cost and simultaneous-detection method, composites of antibody-nano-Au-particles, DON-BSA, FB1-BSA, and AFB1-BSA were prepared to establish a lateral flow assay based on competitive inhibition. The results suggested that the visual detection limits for DON, FB1 and AFB1 were 10, 30, and 10 ng mL−1, respectively. On the other hand, it had no reactivity to T-2 toxin, zearalenone, ochratoxin A and nivalenol, which demonstrates good specificity. Besides, the strip had good repeatability and stability. Therefore, DON, FB1 and AFB1 would be screened simultaneously by lateral flow assay for food safety.

A dual-model “on-super off” photoelectrochemical (PEC)/ratiometric electrochemical (EC) biosensor based on signal enhancing and quenching combining three-dimensional (3D) DNA walker strategy was designed for the ultrasensitive and accurate detection of microRNA-224 (miRNA-224). The “signal on” PEC state was achieved by methylene blue labeled hairpin DNA (MB-DNA) for sensitizing CdS QDs. Then numerous transformational ferrocene labeled DNAs (Fc-DNAs) converted by target-induced 3D DNA walker amplification with the help of Ag nanocubes (NCs) label DNA (Ag-DNA) were introduced to open hairpin MB-DNA. Such configuration change would relocate the sensitizer MB and the quencher Fc, whereas energy transfer placed between Ag NCs and CdS QDs, thereby significantly quenching the PEC signal to obtain “super off” state. Meanwhile, these changes resulted in a decreased oxidation peak current of MB (IMB) and an increased that of Fc (IFc). MiRNA-224 was also detected on basis of the dual-signaling EC ratiometric method for complementary PEC detection. Benefiting from different mechanisms and relatively independent signal transduction, this approach not only avoided interference from difficult assembly but also outstandingly increased sensitivity by distance-controllable signal enhancing and quenching strategies. As a result, the detection ranges of 0.1–1000 fM with a low detection limit of 0.019 fM for PEC, and 0.52 to 500 fM with a low detection limit of 0.061 fM for EC, were obtained for miRNA-224, which opens a new avenue for designing numerous elegant biosensors with potential utility in bioanalysis and early disease diagnosis.

DNA (cytosine-5)-methyltransferase1 (DNMT1) is the most abundant DNA methyltransferase in somatic cells, and it plays an important role in the initiation, occurrence, and rehabilitation of tumors. Herein, we developed a novel strategy for the detection of the level of DNMT1 in human plasma using the self-assembled nucleic acid probe signal amplification technology. In this method, the DNMT1 monoclonal antibody (McAbDNMT1) was immobilized on carboxyl magnetic beads to form immunomagnetic beads and then captured DNMT1 specifically. After that, DNMT1 polyclonal antibody (PcAbDNMT1) and biotinylated sheep anti-rabbit IgG (sheep anti rabbit IgG-Biotin) were sequentially added into the system to react with DNMT1 and form biotinylated double antibody sandwich immunomagnetic beads. In the presence of the bridging medium streptavidin, the biotinylated double antibody sandwich immunomagnetic beads would form a complex with biotinylated poly-fluorescein (Biotin-poly FAM), and the fluorescence intensity of the complex was proportional to the concentration of DNMT1. Immunomagnetic beads can capture the target DNMT1 in the sample, and Biotin-poly FAM can realize signal amplification. Using these strategies, we got a linear range of the system for DNMT1 level detection was from 2 nmol/L to 200 nmol/L, and the limit of detection (LOD) was 0.05 nmol/L. The method was successfully applied for the determination of DNMT1 in human plasma with the recovery of 101.3–106.0%. Therefore, this method has the potential for the detection of DNMT1 level in clinical diagnosis.

An ultrasensitive and selective photoelectrochemical (PEC) biosensor with cathodic background signal was developed for the detection of carcinoembryonic antigen (CEA) based on innovative plasmonic TiO

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.

Highly specific detection of tumor-derived exosomes is of great significance to improve the accuracy of diagnosis of cancer. Herein, based on the proximity hybridization-mediated fluorescence resonance energy transfer (FRET), a novel strategy for highly specific detection of tumor-derived exosomes was proposed by combining detection of multiple exosomal surface markers. Exosomes were enriched and separated by CD63 aptamer-functionalized nanomagnetic beads; a pair of FAM-labeled proximity probes simultaneously bind to EGFR and EpCAM on exosomes surface, which could mediate the formation of a stable DNA self-assemble complex with TAMRA-labeled signal probes through proximity hybridization, and then trigger the FRET between FAM and TAMRA to achieve highly specific detection of exosomes co-expressed CD63/EGFR/EpCAM with a LOD of 400 particles/μL. The relative standard deviation was lower than 7.3% in 50% UC-FBS due to the ratiometric signal improving its anti-interference ability. Importantly, in addition to effectively distinguish exosomes derived from A549 cells and BEAS-2B cells, the feasibility of exosomes detection in human serum was verified, and the level of exosomes co-expressed CD63/EGFR/EpCAM in non-small cell lung cancer patients (n=15) was significantly higher than that of healthy people (n=15) (P

This work aims 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 was a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample.

It is challenging but imperative to merge imaging agents and small molecule therapeutics into one nanoentity for diagnosis and treatment. Herein, we constructed polymeric nanoparticles for targeted delivery and combination chemotherapy, which formed through host-guest interactions among three elements: 1) β-cyclodextrin polymer (poly-β-CD), as the backbone of nanoparticles; 2) two antitumor drugs-doxorubicin (DOX) and docetaxel (DTX); and 3) aptamers labeled with adamantane and fluorescein (Ad-aptamer-FAM), as recognition elements. First, polymeric nanoparticles, termed self-assembled supramolecular nanoparticles (SSNPs), were formulated by combining hydrophobic DTX and DOX with poly-β-CD via host-guest interactions. Then, the surface of SSNPs modified the aptamer to acquire targeting ability; such nanoparticles were termed targeted self-assembled supramolecular nanoparticles (T-SSNPs). As evidenced by MTS assay data, T-SSNPs exhibited significant selective cytotoxicity toward target cells. The results also indicated that combination drugs achieved a good synergistic effect with a combination index of 0.43. Thus, an effective and simple drug delivery system was constructed for targeted delivery and combination chemotherapy.

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.; © 2018 Society of Chemical Industry.

Fei Yu,1,* Ya-min Xiong,1,* Song-cheng Yu,1 Lei-liang He,1 Shan-shan Niu,1 Yu-ming Wu,1 Jie Liu,1 Ling-bo Qu,2 Li-e Liu,1 Yong-jun Wu1 1College of Public Health, 2College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China *These authors contributed equally to this work Background: DNA methyltransferase 1 (DNMT1), a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase) detection have focused on the prokaryote MTase and its activity.Methods: A magnetic fluorescence-linked immunosorbent assay (FLISA) using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized.Results: Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1–1,500 ng/mL, the recovery was 91.67%–106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%–11.29% and 7.03%–11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA) kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r=0.866, p=0.001). The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter than ELISA kits.Conclusion: These indicated that the proposed FLISA method was sensitive and high throughput and can quickly screen the level of DNMT1 in serum samples. Keywords: DNA methyltransferase 1, quantum dots, fluorescence immunoassay, magnetic carboxyl beads, high throughput, serum sample

Self-assembly of building blocks for constructing multifunctional materials has opened prospects for sensing applications in the biomedical fields. In particular, the combination of aptamer with DNA assembly-based nanotechnology has greatly improved the performance of cancer cell detection. Nevertheless, the cancer cell detection strategies of integrating aptamer with protein are relatively sparse. So we have developed a self-assembled aptamer method to realize the efficient capture and rapid detection of cancer cells by ingeniously combining aptamer modified magnetic nanoparticles as capture nanoprobes with self-assembled aptamer/protein hybrid probes (SAPPs) as signal amplification probes. By merely mixing the component materials together simultaneously, the SAPPs, integrating aptamer for cancer cell recognition with protein for amplifying signal, were fabricated by DNA-governed one-step assembly. In addition, the SAPPs-based method exhibits efficient capture, rapid (about 45 min) and specific CCRF-CEM detection performance, with limits of detection down to 75 cells/mL in buffer and 200 cells/mL in whole blood.

An ultrasensitive and selective photoelectrochemical (PEC) biosensor with cathodic background signal was developed for the detection of carcinoembryonic antigen (CEA) based on innovative plasmonic TiO2@Au nanoparticles//CdS quantum dots (TiO2@Au NPs//CdS QDs) photocurrent-direction switching system, coupling with hybridization chain reaction (HCR) for the signal amplification. Firstly, innovative TiO2@Au NPs were successfully fabricated through in situ ascorbic acid-reduction of Au NPs dispersed on TiO2 surface, and TiO2@Au NPs as the photoactive material showed a cathodic background signal. When target CEA existed, a sandwich-type reaction was performed in capture CEA aptamer-modified TiO2@Au NPs and trigger CEA aptamer. Interestingly, after HCR triggered by target CEA, a mass of CdS QDs were introduced into the biosensing platform, resulting in the formation of TiO2@Au NPs//CdS QDs system, along with the switch of photocurrents from cathodic to anodic. The obtained remarkable anodic photocurrent was depended on the localized surface plasmon resonance (LSPR) effect of Au between TiO2 and CdS. Under the optimal conditions, plasmonic TiO2@Au NPs//CdS QDs photocurrent-direction switching PEC biosensing platform with cathodic background signal exhibited ultrasensitive for the determination of CEA with a low limit of detection of 18.9 fg/mL. Importantly, the proposed PEC biosensor can eliminate the interferences of the initial photocurrent and background signal, and has high-efficiency anti-interference ability, satisfactory stability and excellent reproducibility, which may have great potentials in bioanalysis and disease diagnosis.

An idea drug carrier, with good binding affinity, selectivity, drug payload capacity, and cellular internalized capability, will greatly improve the efficiency of target delivery. Herein a self-assembled and multivalent DNA nanostructure was developed as drug carrier for efficient and targeted delivery. The DNA structure was similar to that of a centipede, composed of trunk and legs: The trunk was a self-assembled DNA scaffold via hybridization chain reaction (HCR) from two biotinylated hairpin monomers created upon initiation by a trigger DNA, and the legs were biotinylated aptamers conjugated to the trunk via streptavidin-biotin affinity interaction. The long trunk of the "DNA nanocentipede" was loaded with doxorubicin (Dox), and the legs were SMMC-7721 cell-binding aptamers (Zy1) which functioned as targeting moieties to firmly and selectively grasp target cells. The results of agarose gel electrophoresis and fluorescence anisotropy confirmed that Zy1-based DNA nanocentipedes (Zy1-Nces) were successfully constructed. Flow cytometric analyses demonstrated that Zy1-Nces were more effective than free Zy1 in binding affinity and selectivity due to a multivalent effect. Confocal microscopy studies demonstrated that the internalization was highly dependent on the higher valences of DNA nanocentipedes without the loss of selectivity. Meanwhile, Zy1-Nces exhibited high drug-loading capacity and selective drug transport. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed enhanced cellular cytotoxicity of the Dox-loaded Zy1-Nces (Zy1-Nces-Dox) to the target SMMC-7721 cells but not negative control L02 cells. This approach is applicable to prepare drug carriers for other targets by construction of the nanocentipedes with relevant nucleic acid fragments.

The building of artificial systems with similar structure and function as cellular compartments will expand our understanding of compartmentalization related biological process and facilitate the construction of biomimetic highly functional structures. Herein, surface phenylboronic acid functionalized mesoporous silica sphere was developed as a biomimetic dopamine gated compartment, in which the ionic permeability can be well modulated through the dopamine-binding induced charge reversal. As the phenylboronic acid is negatively charged, the negatively charged 1, 3, 6, 8-pyrenetetrasulfonic acid (TPSA) was hindered from permeation into the biomimetic compartment. However, the presence of dopamine and its binding with phenylboronic acid reversed the gatekeeper shell from negative to positive charged and gated the permeation of TPSA into the interior. The dopamine gated permeation phenomenon resembles that in biological system, and thus the phenylboronic acid functionalized mesoporous silica sphere was taken as a simple model for dopamine gated ion channel decorated biological compartment. It will also contribute to the development of artificial cell and responsive nanoreactor.

A novel enzyme-free amplified surface plasmon resonance (SPR) biosensor for microRNA (miRNA) detection was developed based on gold nanoparticles (AuNPs) coupled with DNA supersandwich. In the detection strategy, the DNA-linked AuNPs as the primary amplification element, not only hybridized with the capture DNA on the Au film to amplify SPR signal but also initiated the subsequent secondary amplification, i.e. DNA supersandwich formation of two report probes. In the presence of target, stem–loop structure of capture DNA on the Au film surface was unfolded, and DNA-linked AuNPs were bound to Au film by hybridization with terminus of capture DNA. Then, the carried assistant DNA on the AuNPs could trigger an alternative hybridization reaction of two report probes, resulting in the formation of DNA supersandwich. Due to the electronic coupling between localized plasmon of AuNPs and the surface plasmon wave associated with Au film, as well as the enhancement of the refractive index of the medium next to the metal film caused by DNA supersandwich structure, the shift of resonance angle was enhanced obviously. By employing the enzyme-free dual signal amplification strategies, as low as ca. 8 fM miRNA-21 could be detected. Moreover, this assay also showed high selectivity toward single-base mismatch, and demonstrated its applicability for the target detection in human serum. This work may provide great potential applications in future clinical analysis.

Convenient and rapid detection of Cu2+ in serum has important clinical significance for related diseases. Herein, a ratiometric detection of Cu2+ based on specific short peptide (SPGH)-assisted enhanced chemiluminescence resonance energy transfer (CRET) has been constructed. An interesting phenomenon that tetra-peptide with the sequence of Ser-Pro-Gly-His (SPGH) was able to specifically and remarkably enhance the catalytic performance of Cu2+ has been discovered, the enhancement mechanism is mainly due to the significant increasing generation of Cu2+-induced hydroxyl radical after addition of SPGH. The chemiluminescence (CL) of the luminol-H2O2 system was efficiently catalyzed by the formed Cu(SPGH)2 complex, which acts as an internal light source to triggers on the fluorescence of QDs via CRET, allowing the ratiometric detection of Cu2+ within 15 min with improved selectivity and sensitivity. The relative standard deviation was lower than 6.38 % due to the ratiometric signal improving its anti-interference ability. Importantly, it is feasible to detect total Cu in human serum, and the results were consistent with that of ICP-AES (r = 0.9321, n=55). The reliable, rapid, yet simple ratiometric detection of Cu2+ in serum based on SPGH-assisted enhanced CRET provides a promising potential alternative method for Cu2+ detection in clinical application.

In this paper, a label-free fluorescence nanoprobe is constructed based on poly(thymine) single strand DNA-templated Copper nanocluster (denote as: T-CuNCs) for the detection of hydrogen peroxide. In the assay, the fluorescent T-CuNCs will generate though the reaction of Cu2+, poly(thymine) and sodium ascorbate. However, the hydroxyl radical (.OH) will generated in the presence of H2O2, which is able to induced the oxidative lesions of poly(thymine) single chain DNA and lead to the poly(thymine) being splitted into shorter or single oligonucleotide fragments and lose the ability to template the fluorescent T-CuNCs again. Therefore, H2O2 can be detected by monitoring the fluorescence strength change of T-CuNCs. The experimental results show that the fluorescence intensity change of T-CuNCs has fantastic linearity versus H2O2 concentration in the range of 1–30 μM (R2 = 0.9947) and 30–80 μM (R2 = 0.9972) with the limit of detection (LOD) as low as 0.5 μM (S/N = 3). More important, the fluorescent nanoprobe was also successfully utilized on the detection of H2O2 in serum samples. Therefore, a label-free, costless and effective fluorescence method has been established for the detection of H2O2, the intrinsic properties of the nanoprobe endow its more potential applications in chemical and biological study.

Early detection of cancer is of great significance for disease prevention and diagnosis. However, the levels of most cancer markers are quite low in the early stages of disease, so it is urgent to develop a highly sensitive detection method. In this study, a label-free and highly sensitive colorimetric strategy was developed for the detection of the vascular endothelial growth factor165 (VEGF165) in human serum. First, a convenient biosensor was constructed by immobilizing VEGF165 on a microplate, where aptamers bound with VEGF165 to form a complex. Then, streptavidin labeled-horseradish peroxidase (HRP-SA) combined with the complex via the interaction between streptavidin and biotin, thus catalyzing the 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 system to produce colored products. In the presence of target, immobilized VEGF165 and target competitively bound with the aptamers, resulting in a reduction of the colorimetric signal. Moreover, the optical density (OD) signal decreased with the increase of target concentration. The strategy showed a broad linear range (0.1–100 ng/mL) and a rather low detection limit of 10 pg/mL with good precision and selectivity. Further, the proposed method was successfully applied in detecting VEGF165 in human serum. The detection results of serum samples showed that the proposed assay had a high correlation with CLEIA kits (r = 0.971, P = 0.001). It has potential for application in clinical research and diagnosis.

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 MβCD 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.

Deoxynivalenol (DON), Fumonisin B

The detection of nucleic acids is fundamental for studying their functions and for the development of biological studies and medical diagnostics. Herein, we report a new strategy for nucleic acid amplified detection by combining target-catalyzed dynamic assembly with host-guest interaction between β-cyclodextrin polymer (β-CDP) and pyrene. In this strategy, a metastable pyrene-labeled hairpin DNA probe (probe H1) and a metastable unlabeled hairpin DNA probe (probe H2) were elaborately designed as the assembly components, which were kinetically handicapped from cross-opening in the absence of target DNA. In this state, pyrene labled at the 5'-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to significant fluorescence enhancement. Once the dynamic assembly was catalyzed by target DNA, a hybridized DNA duplex H1-H2 would be created continuously. In this state, it is difficult for pyrene to enter the cavity of β-CDP due to steric hindrance and weak-binding interaction, leading to a weak fluorescent signal. Thus, target DNA could be detected by this simple mix-and-detect amplification method without the need of expensive and perishable protein enzymes. As low as 10 pM of the target DNA was detected by this assay, which was comparable to that of some reported enzyme-dependent amplification methods. Meanwhile, the proposed method was further successfully applied to detect DNA in cell lysate samples, showing great potential for target detection from complex fluids. In addition, as a novel transformation of dynamic DNA assembly technology into enzyme-free signal-amplification analytical application, the proposed strategy has shown great potential for applications in a wide range of fields, such as aptamer-based non-nucleic acid target sensing, biomedicine and bioimaging.

We developed a novel homogeneous fluorescence analysis based on a novel competitive host-guest interaction (CHGI) mechanism between β-cyclodextrin polymer (polyβ CD) and pyrene-labeled probe for biochemical assay. Pyrene labeling with oligonucleotide strands can be recruited and reside in lipophilic cavities of polyβ 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 polyβ CD. The release of pyrene from polyβ 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 polyβ 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.

In the present study, a novel highly sensitive magnetic enzyme chemiluminescence immunoassay (MECLIA) was developed to detect fumonisin BThe 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.

A one-step and facile strategy toward the construction of multicolor polymers via supramolecular self-assembly was proposed. Multicolor polymers were simply prepared by the self-assembly of adamantane-labeled fluorescein, adamantane-labeled rhodamine B and β -cyclodextrin polymers via host–guest interaction between β -cyclodextrin and adamantane. Multicolor polymers showed several interesting properties: multiple emission signatures by a single wavelength excitation, easy tunability, intense fluorescence, high photostablility. In addition, the self-assembly approach implied a facile and flexible strategy for constructing functionalized materials, such as multicolor materials for biological labeling and imaging, and sensing materials for the detection of physiological parameters.

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

A novel method for the detection of PDGF-BB has been developed using double-strand DNA-copper nanoparticles (dsDNA-CuNPs) as fluorescent markers. This assay relies on the premise that the aptamer-based probe undergoes a conformational change upon binding with target protein, and subsequently triggers polymerization reaction to generate dsDNA. Then, the resultant dsDNA can be used as a template for the formation of CuNPs with high fluorescence. Under the optimized conditions, the proposed assay allowed sensitive and selective detection of PDGF-BB with a detection limit of 4 nmol/L. This possibly makes it an attractive platform for the detection of a variety of biomolecules whose aptamers undergo similar conformational change.

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.

It was difficult to detect small molecules directly using conventional surface plasmon resonance (SPR) biosensors since the changes of refractive index, which was resulted by binding small molecules, were usually small. In this paper, split aptamer fragments were used for the construction of SPR biosensor to determine small molecule such as adenosine with high sensitivity. An aptamer for adenosine was designed to be two flexible ssDNA pieces, one was tethered on Au film and the other was modified on Au nanoparticles (AuNPs). In the presence of adenosine, two ssDNA pieces reassembled into the intact aptamer structure and the AuNPs-labeled adenosine–aptamer complex was formed on the Au film. Then, the resonance wavelength shift was enhanced obviously, due to the electronic coupling between the localized plasmon of AuNPs and the surface plasmon wave associated with Au film. The results confirmed that this biosensor could detect adenosine with high sensitivity and selectivity. The limitation of detection (LOD) of this SPR biosensor was ca. 1.5 pM, which was an approximately ca. 2–3 order of magnitude lower than that of those SPR biosensors which utilized competitive methods.

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.

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 β-cyclodextrin polymer (poly-β-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.

A strategy for T4 polynucleotide kinase activity detection was proposed based on a β-cyclodextrin polymer (polyβ-CD) and an exonuclease reaction. The fluorescence of pyrene enhanced by more than 10 times in the presence of polyβ-CD, and a simple detection of T4 PNK was achieved with a detection limit of 0.02 units per mL.

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. β-cyclodextrin polymer (βCDP)-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)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP 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)/βCDP 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/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/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/βCDP@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/βCDP@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/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

A novel two-photon absorption (TPA) nanomicelle through the “host–guest” chemistry has been developed and successfully applied in tumor tissue imaging in this work.

We present a novel strategy for construction of a conformational switch of a molecular beacon based on the combination of nucleic acid (DNA) self-assembly and reversible host–guest inclusion interaction. With the functionalized probe, the nucleic acid hybridization process can be easily controlled with a photoactivated and thermal regeneration mode.

A single labeled fluorescence probe was designed based on the efficient quenching ability of a G-quadruplex instead of traditional quenchers. Using this probe, we have investigated the quenching ability of a G-quadruplex and developed a new approach to analyze mercury ions and cysteine with high specificity and sensitivity. Owing to the high quenching ability of the G-quadruplex and the fidelity of the “thymine–Hg2+–thymine” binding motif, this approach can detect 4.0 nM mercury ions, which is lower than the 10 nM US EPA limit in drinking water.

Based on the "Y" junction structure and restriction endonuclease-aided target recycling strategy, an electrochemical biosensor for DNA detection was developed. This universal biosensor was suitable for detecting different sequences of target DNA by changing the sequence of capture and assistant strands.