Microfluidic paper-based chemiluminescence sensing platform based on functionalized CaCO3 for time-resolved multiplex detection of avian influenza virus biomarkers.

Multiplex detection can enhance diagnostic precision and improve diagnostic efficiency, providing important assistance for epidemiological investigation and epidemic prevention. There is a great need for multi-detection sensing platforms to accurately diagnose diseases. Herein, we reported a µPAD-based chemiluminescence (CL) assay for ultrasensitive multiplex detection of AIV biomarkers, based on three DNAzyme/Lum/PEI/CaCO3. Three time-resolved CL signals were sequentially generated with detection limits of 0.32, 0.34, and 0.29 pM for H1N1, H7N9, and H5N1, respectively, and with excellent selectivity against interfering DNA. The recovery test in human serum displayed satisfactory analysis capabilities for complex biological samples. The µPAD-based CL assay achieved multiplex detection within 70 s, with a high time resolution of 20 s. The proposed strategy has the advantages of low cost, high sensitivity, good selectivity, and wide time resolution, the µPAD-based CL assay has shown great potential in the early and accurate diagnosis of diseases.

Tm3+ mediated multicolor luminescence of NaYbF4:Er,Tm@NaYF4 for advanced anti-counterfeiting.

Lanthanide doped multicolor luminescent materials have attracted extensive attention due to their advanced anti-counterfeiting properties. However, designing a simple, hard-to-copy and multicolor anti-counterfeiting strategy based on upconversion nanoparticles (UCNPs) remains a huge challenge. Herein, a strategy to modulate luminescence color by altering the mediating action of Tm3+ was proposed. As a proof of concept, the mediating action of Tm3+ was explored in NaYbF4:30%Er,1%Tm@NaYF4 by changing the doping ratio of Yb3+/Er3+/Tm3+, and red, yellow and blue luminescence was successfully obtained. Then, NaYbF4:x%Er,1%Tm@NaYF4 (x = 2, 10, 30, 50, 99), NaYbF4:x%Er@NaYF4 (x = 2, 10, 30, 50, 100) and NaYbF4:1%Tm@NaYF4:x%Er@NaYF4 (x = 2, 10, 30, 50, 100) were synthesized to further identify that the mediating action of Tm3+ was related to the doping ratio and distance between dopant ions. In addition, the luminescence color of NaYbF4:30%Er,1%Tm@NaYF4 changed from red to yellow with the increase of excitation power density. Based on the above, NaYbF4:Er,Tm@NaYF4 UCNPs show excellent performance in anti-counterfeiting of paintings, thus revealing their great potential in advanced anti-counterfeiting applications.

Microbiome-friendly PS/PVP electrospun fibrous membrane with antibiofilm properties for dental engineering.

Dental caries is one of the most prevalent and biofilm-associated oral diseases in humans. Streptococcus mutans, with a high ability to form biofilms by adhering to hard surfaces, has been established as an important etiological agent for dental caries. Therefore, it is crucial to find a way to prevent the formation of cariogenic biofilm. Here, we report an electrospun fibrous membrane that could inhibit the adhesion and biofilm formation of S. mutans. Also, the polystyrene (PS)/polyvinyl pyrrolidone (PVP) electrospun fibrous membrane altered the 3D biofilm architecture and decreased water-insoluble extracellular polysaccharide production. Notably, the anti-adhesion mechanism which laid in Coulomb repulsion between the negatively charged PS/PVP electrospun fibrous membrane and S. mutans was detected by zeta potential. Furthermore, metagenomics sequencing analysis and CCK-8 assay indicated that PS/PVP electrospun fibrous membrane was microbiome-friendly and displayed no influence on the cell viability of human gingival epithelial cells and human oral keratinocytes. Moreover, an in vitro simulation experiment demonstrated that PS/PVP electrospun fibrous membrane could decrease colony-forming unit counts of S. mutans effectively, and PS/PVP electrospun fibrous membrane carrying calcium fluoride displayed better anti-adhesion ability than that of PS/PVP electrospun fibrous membrane alone. Collectively, this research showed that the PS/PVP electrospun fibrous membrane has potential applications in controlling and preventing dental caries.

Highly Sensitive Detection of T790 M with a Three-Level Characteristic Current by Thymine-Hg(II)-Thymine in the α-Hemolysin Nanopore.

Sensitive detection of resistance mutation T790 M is of great significance for early diagnosis and prognostic monitoring of non-small-cell lung cancer (NSCLC). In this paper, we showed a highly sensitive detection strategy for T790 M using a three-level characteristic current signal pattern in an α-hemolysin nanopore. A probe was designed that formed a C-T mismatched base pair with wild-type/P and a T-T mismatched with the T790M/P. The T790M/P produced a unique three-level characteristic current signal in the presence of mercury ions(II): first, T790M-Hg2+-P entering the vestibule of α-HL under the transmembrane potential and overhang of probe occupying the ß-barrel, then probe unzipping from the T790M/P, T790 M temporally residing inside the nanocavity due to the interaction with Hg(II), and finally T790 M passing through the ß-barrel. The blocking current distribution was concentrated with a small relative standard deviation of about 3%, and the signal peaks of T790 M and wild-type can be completely separated with a high separation resolution of more than 2.5, which achieved the highly sensitive detection of T790 M down to 0.001 pM (confidence level P 95%) with a linear range from 0.001 pM to 1 nM in human serum samples. This highly sensitive recognition strategy enables the detection of low abundance T790 M and provides a method for prognostic monitoring in NSCLC patients.

Multifunctionalized flower-like gold nanoparticles with high chemiluminescence for label-free sensing of the hepatitis C virus core protein.

Developing functionalized nanomaterials with strong chemiluminescence (CL) properties is highly significant for ultrasensitive bioanalysis. Here, we report chitosan (CS), luminol, and Co2+-functionalized flower-like gold nanoparticles (Co2+/CS/Lum/AuNFs) with strong CL for the label-free sensing of the HCV core protein (HCVcp). The Co2+/CS/Lum/AuNFs exhibited a greatly enhanced CL emission at around 425 nm, which is 50 times stronger than that of CS/Lum/AuNFs, and is superior to other commonly reported CL nanomaterials. The HCVcp aptamer (HCVcp-apt) further functionalized the surface of the Co2+/CS/Lum/AuNFs through electrostatic interactions blocked the Co2+ catalytic site, depressing the CL. Owing to the high affinity of HCVcp for the HCVcp-apt, the presence of HCVcp predominated its binding and effectively separated the HCVcp-apt from the surface of the Co2+/CS/Lum/AuNFs, so that the CL intensity was significantly enhanced. As the results showed, the HCVcp-apt/Co2+/CS/Lum/AuNFs were successfully used to detect the HCVcp in human serum samples with a linear range from 0.50 ng mL-1 to 1.00 µg mL-1, a detection limit of 0.16 ng mL-1 and an excellent selectivity over other analogs. The strategy is universal for the development of the ultrasensitive detection of other proteins in the field of early disease diagnostics.

High-fidelity biosensing of dNTPs and nucleic acids by controllable subnanometer channel PaMscS.

Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1-3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.

Current Simultaneous Discrimination of Mismatched MicroRNAs Using Base-Flipping within the α-Hemolysin Latch.

The simultaneous discrimination of let-7 microRNAs (miRNAs) would greatly facilitate the early diagnosis and prognosis monitoring of diseases. In this work, a molecular beacon DNA probe was designed to be able to flip out its mismatched cytosine base within the α-hemolysin (α-HL) latch and generate completely separated blocking currents to identify the single-base difference. As a result, the characteristic blocking current of fully matched MB/let-7a and single-base mismatched MB/let-7f was 84.30 ± 0.92 and 87.05 ± 0.86% (confidence level P 95%), respectively. Let-7 miRNA family let-7a and let-7f were completely simultaneously discriminated, which could be attributed to the following strengths. (1) The statistic distribution of blocking current is extremely concentrated with a small relative standard deviation (RSD) of less than 1% and a narrow distribution range. (2) Complete separation is achieved with a high separation resolution of 1.54. (3) The cytosine base flipping out within the α-HL latch provides a universal labeling-free strategy to simultaneously discriminate the single-base mismatch. Overall, the target let-7f sequences were detected with a linear range from 0.001 to 10 pM in human serum samples containing 200 nM let-7a. Great potential has been demonstrated for precise detection, early diagnosis, and prognosis monitoring of diseases related to single-base difference.

One-step fast and label-free imaging array for multiplexed detection of trace avian influenza viruses.

Rapid and low-cost diagnosis of multiple infectious diseases is of great significance especially in densely populated or resource-constrained settings. Herein, we developed a one-step fast and label-free imaging array for multiplexed detection of trace avian influenza virus (AIV) DNA biomarkers. By designing a series of specific and efficient catalytic hairpin assembly (CHA) amplification reactions and utilizing thioflavin T, a specific G-quadruplex fluorescence probe, three subtypes of AIV DNA biomarkers (H1N1, H7N9 and H5N1) were simultaneously and quickly detected within only 20 min, which just needed a small reagent volume of 50 µL and a smartphone instead of a spectrometer. With the combination of fluorescence imaging output and grey-level analysis, the array sensor can be on-site with the limit of detection of 136 pM, 141 pM and 129 pM for H1N1, H7N9 and H5N1, respectively. The imaging array also displayed good mismatch discrimination, excellent anti-interference, and real sample application. In view of its advantages of fast detection, low cost and multiplexed analysis, the imaging array is expected to have potential applications for early infectious disease diagnosis in resource-constrained settings.

Electrochemiluminescent Chiral Discrimination with a Pillar[5]arene Molecular Universal Joint-Coordinated Ruthenium Complex.

A bicyclic pillar[5]arene derivative fused with a bipyridine side ring, a so-called molecular universal joint (MUJ), was synthesized, and the pair of enantiomers was resolved by high-performance liquid chromatography enantioresolution. The electrochemiluminescent detection based on the ruthenium complex of the enantiopure MUJ showed excellent chiral discrimination toward certain amino acids.

A label-free fluorescent biosensor based on a catalyzed hairpin assembly for HIV DNA and lead detection.

Herein, a label-free fluorescent signal amplification system based on a catalyzed hairpin assembly (CHA) is reported. In this system, two hairpin probes, H1 and H2, were well-designed in which G-quadruplex sequences were integrated into H2. The CHA reaction was triggered by target/trigger DNA and G-quadruplex sequences were released, which can bind the fluorescent amyloid dye thioflavin T (ThT) to provide fluorescence signals. At the same time, target/trigger DNA was released from the product of the CHA reaction (H1-H2), which continued to initiate the next CHA cycle, and the signal was eventually amplified. This signal amplification approach has been successfully used to develop a label-free fluorescent sensing platform for sensitive detection of human immunodeficiency virus (HIV) DNA and Pb2+.

Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell.

Interface between neuron cells and biomaterials is the key to real-time sensing, transmitting and manipulating of neuron activities, which are the long-term pursue of scientists and gain intense research focus recently. It is of great interest to develop a sensor with exquisite sensitivity and excellent selectivity for real-time monitoring neurotransmitters transport through single live cell. Sensing techniques including electrode-based methods, optogenetics, and nanowire cell penetration systems have been developed to monitor the neuron activities. However, their biocompatibilities remain a challenge. Protein nanopores with membrane compatibility and lumen tunability provide real-time, single-molecule sensitivities for biosensing of DNA, RNA, peptides and small molecules. In this study, an engineered protein nanopore MspA (Mycobacterium smegmatis porin A) through site-directed mutation with histidine selectively bind with Cu2+ in its internal lumen. Chelation of neurotransmitters such as L-glutamate (L-Glu), dopamine (DA) and norepinephrine (NE) with the Cu2+ creates specific current signals, showing different transient current blockade and dwell time in single channel electrophysiological recording. Furthermore, the functionalized M2MspA-N91H nanopores have been embedded in live HEK293T cell membrane for real-time, in situ monitoring of extracellular L-glutamate translocating through the nanopore. This biomimetic neurotransmitter nanopore has provided a new platform for future development of neuron sensors, drug carrier and artificial synapse.

Rapid and colorimetric detection of nucleic acids based on entropy-driven circuit and DNAzyme-mediated autocatalytic reactions.

In this work, a novel, rapid and enzyme-free colorimetric biosensor for the detection of nucleic acids has been developed based on entropy-driven (EDC) circuit and DNAzyme-mediated autocatalytic reactions. On sensing the target DNA, the EDC reaction could be initiated and the intact Mg2+-dependent DNAzyme was formed in the reaction product; then, a "mimic target" DNA was generated during the cleavage process of DNAzyme, which in turn catalyzed the EDC reaction corresponding to an autocatalytic process. Meanwhile, numerous G-quadruplex structures were released and further interacted with hemin to form peroxidase-mimicking DNAzyme, inducing a remarkably amplified colorimetric signal. This autocatalytic EDC (AEDC) sensing system exhibited a linear relationship in the range from 20 pM to 10 nM with a detection limit of 10.2 pM. More importantly, benefitting from the Mg2+-dependent DNAzyme-mediated autocatalytic reaction, the detection time (20 min) was significantly reduced compared to that for the reported EDC strategies. In addition, this sensing system has been applied for the detection of target DNA in human serum samples, indicating that it is promising for the on-site and real-time detection of nucleic acids in biomedical research and disease diagnosis.

Detection of Circulating Tumor Cells in Breast Cancer Patients by Nanopore Sensing with Aptamer-Mediated Amplification.

Circulating tumor cells (CTCs) have been utilized in the diagnosis and prognosis of tumor. However, the CTC concentration is extremely low to be detected in peripheral blood. Many existing methods suffer from either expensive labeling or complex operation. In this study, we constructed a label- and enzyme-free and sensitive method to detect the breast cancer CTCs. First of all, a probe containing a breast cancer cell-specific aptamer and a complementary single-stranded DNA (trigger DNA P1) were designed. When the target cells are present, the aptamer binds to the CTCs and releases P1 which triggers the strand displacement amplification. This process generates three-way junction structure DNA, the specific translocation signals of which are identified by nanopore assay. The detection limit of tumor cells is 5 in the current experimental setup and can be further reduced. Furthermore, the method is demonstrated in a clinical sample test with high recovery rate and accuracy. Our results suggest that this method could be applied to early diagnosis of metastatic recurrence and prognosis determination.

Early Monitoring Drug Resistant Mutation T790M with a Two-Dimensional Simultaneous Discrimination Nanopore Strategy.

With the aim of detecting low frequency of drug resistant mutation T790M against wild-type sequences, we reported a two-dimensional signal analysis strategy by combining a three locked nucleic acids (LNAs)-modified probe (LP15-3t) and an α-HL nanopore. The specific hybridization of the LP15-3t probe with the T790M generated unique long two-level signals, including characteristic blocking current and characteristic dwell time. Due to the significant dwell time difference (114.2-fold) and the blocking current difference ranging from 81% to 96%, this two-dimensional signal analysis strategy can simultaneously distinguish T790M sequences with a sensitivity of 0.0001% against wild-type sequences. The LOD of T790M was 0.1 pM. This high discrimination capability would have great potential in the detection of rare mutation sequences and the early monitoring of clinical outcome of NSCLC patients with TKI drug resistance.

An enzyme-free and label-free visual sensing strategy for the detection of thrombin using a plasmonic nanoplatform.

An enzyme-free and label-free visual sensing strategy was developed for sensitively detecting thrombin using a plasmonic nanoplatform. Both the thrombin-triggered catalytic hairpin assembly (CHA) amplification reaction and G-quadruplex/hemin DNAzyme-controlled plasmonic signal readout were engineered on an electrospun nanofibrous membrane. Owing to its large specific surface area and porous structure, the nanofibrous membrane enhanced the loading capacity of B-H2 and the interface interaction efficiency. This plasmonic nanoplatform was used to perform the sensitive and naked-eye detection of thrombin as low as 1.0 pM in human serum samples. This visual strategy can discriminate thrombin from other co-existing proteins very well. Moreover, the visual sensing platform exhibited excellent reusability and long-term stability. The proposed enzyme-free and label-free plasmonic nanoplatform is low-cost, easy to operate and highly sensitive, and has potential applications in the point-of-care detection of protein biomarkers.

Demethyleneberberine attenuates concanavalin A-induced autoimmune hepatitis in mice through inhibition of NF-κB and MAPK signaling.

Demethyleneberberine (DMB) is a natural product which has been reported to possess mitochondria-targeting anti-oxidative and anti-inflammatory effect. However, the pharmacological action and molecular mechanism of DMB on autoimmune hepatitis (AIH) have not been explored. In this study, AIH was induced by intravenously injecting Con A (20 mg/kg) in mice for 8 h, and DMB protected against Con A-induced AIH, evidenced by obvious reduction of hepatic enzymes in serum and histological lesion. DMB significantly inhibited the infiltration of CD4+ T cell and Kupffer cell as well as the expression of inflammatory cytokines, such as TNF-α, IL-6, IL-1ß and IFN-γ by ELISA and qPCR analysis. Western blotting analysis illustrated that DMB remarkably inhibited Con A-induced phosphorylation of IKK, IκB, NF-κB p65, ERK, JNK, p38 MAPK and STAT3 induced by Con A. Moreover, DMB also effectively suppressed hepatic oxidative stress with reduction of MDA and elevation of GSH. Taken together, our findings indicated that DMB could prevent Con A-induced AIH by regulating NF-κB and MAPK signaling, suggesting that DMB can serve as a promising candidate for therapy of AIH.

Plasmonic nanoplatform for point-of-care testing trace HCV core protein.

Early diagnosis of hepatitis C virus (HCV) infection is still urgently desired as there is a global healthy burden and no vaccine available. In this work, a plasmonic nanoplatform was engineered with catalytic hairpin assembly (CHA) amplification reaction specifically of HCV core protein (HCVcp), G-quadruplex/hemin DNAzyme and nanofibrous membrane together. HCVcp was detected in whole serum at the ultralow concentration of 1.0 × 10-4 pg/mL with naked eye. By testing serum samples from 30 donors with different viral loads, detection sensitivity of the plasmonic nanoplatform turned out to be much better than that of the commercial ELISA kit. In addition, the plasmonic nanoplatform exhibited high specificity, excellent reusability and long-term stability. Naked-eye detection based on the plasmonic nanoplatform is expected to have potential applications in point-of-care testing (POCT) and early diagnosis of hepatitis C and other infectious diseases.

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

One-step sensitive thrombin detection based on a nanofibrous sensing platform.

Convenient and time-saving one-step strategies for detecting ultralow concentrations of protein biomarkers play key roles in rapid disease diagnosis. In this study, we report a one-step detection method based on a nanofibrous sensing platform via the combination of proximity-induced DNA strand displacement (PiDSD), catalytic hairpin assembly (CHA) amplification and thioflavin T (ThT) binding. The interface behaviors on the nanofibrous membrane were studied to promote interface reaction kinetics and thermodynamics. Thrombin was used as a model biomarker, and the nanofibrous sensing platform achieved a limit of detection as low as 1.0 pM, a wide linear range of 50 pM to 5 nM, excellent specificity and good long-term stability. Compared with previous one-step thrombin detection methods, our one-step detection method is label-free, convenient and much more sensitive; it has potential applications for protein detection in point-to-care testing (POCT) and early diagnosis.

Self-Replication-Assisted Rapid Preparation of DNA Nanowires at Room Temperature and Its Biosensing Application.

A rapid room-temperature DNA nanowires preparation strategy on the basis of self-replicating catalyzed hairpin assembly (SRCHA) was reported. In this system, three hairpin probes (P1, P2, and P3) were well-designed and partially hybridize to each other, and two split trigger DNA sequences were integrated into P1 and P3, respectively. When the SRCHA was initiated by the trigger DNA, a series of DNA assembly steps based on the toehold-mediated DNA strand displacement were activated, and the Y shaped DNA (P1-P2-P3) was formed. In that case, the two split trigger DNA sequences will come into close-enough proximity to form the trigger DNA replicas, which can initiate the additional SRCHA reaction cycles for DNA nanowire preparation, and eventually a rapid room-temperature DNA nanowires preparation strategy without need of fuel strands was successfully developed. Furthermore, the prepared DNA nanowires have been used to develop a rapid and signal amplified sensing platform for sensitive adenosine triphosphate (ATP) detection.