Ultra-Sensitive Portable Visual Paper-Based Viral Molecularly Imprinted Sensor without Autofluorescence Interference.

Detection of the virus is the primary factor to discover and block the occurrence and development of the virus epidemic. Here, an ultrasensitive paper-based virus molecular imprinting sensor is developed to detect two viruses simultaneously in which the detection limit of the influenza virus (H5N1) is 16.0 aM (9.63 × 103 particles/mL) while that of the Hepatitis B Virus (HBV) is 129 fM (7.77 × 107 particles/mL). This paper-based sensor is low cost and is easy to cut, store, and carry. In addition, the visual semiquantitative detection of two viruses is achieved by using two aptamer-functionalized persistent luminescent nanoparticles as signal probes. These probes and the imprinted cavities on the paper-based material formed sandwich-type double recognition of the target viruses. This sensor has extremely high sensitivity to the H5N1 virus, which is of great value to solve the influenza epidemic with the most outbreaks in history, and also opens up a new way for the prevention and control of other virus epidemics. This cheap and portable visual sensor provides the possibility for self-service detection and can greatly reduce the pressure on medical staff and reduce the risk of virus infection caused by the concentration of people to be tested.

A novel heterozygous missense variant of the ARID4A gene identified in Han Chinese families with schizophrenia-diagnosed siblings that interferes with DNA-binding activity.

ARID4A plays an important role in regulating gene expression and cell proliferation. ARID4A belongs to the AT-rich interaction domain (ARID)-containing family, and a PWWP domain immediately precedes its ARID region. The molecular mechanism and structural basis of ARID4A are largely unknown. Whole-exome sequencing (WES) revealed that a novel heterozygous missense variant, ARID4A c.1231 C > G (p.His411Asp), was associated with schizophrenia (SCZ) in this study. We determined the crystal structure of the PWWP-ARID tandem at 2.05 Å, revealing an unexpected mode in which ARID4A assembles with its PWWP and ARID from a structural and functional supramodule. Our results further showed that compared with the wild type, the p.His411Asp ARID mutant protein adopts a less compact conformation and exhibits a weaker dsDNA-binding ability. The p.His411Asp mutation decreased the number of cells that were arrested in the G0-G1 phase and caused more cells to progress to the G2-M phase. In addition, the missense mutation promoted the proliferation of HEK293T cells. In conclusion, our data provide evidence that ARID4A p.His411Asp could cause a conformational change in the ARID4A ARID domain, influence the DNA binding function, and subsequently disturb the cell cycle arrest in the G1 phase. ARID4A is likely a susceptibility gene for SCZ; thus, these findings provide new insight into the role of ARID4A in psychiatric disorders.

Construction of benzoheterocycles by the reaction of α-arylglyoxylic acids and ortho-functionalized aniline under mild and minimal conditions.

This work describes an environmentally friendly method for the synthesis of benzoxazinones, quinoxalinones and benzothiazoles by the reaction of α-arylglyoxylic acids and ortho-functionalized aniline. In this reaction, no other reagents are needed except for reactants and solvents. The reaction was carried out at a mild temperature of 50 °C with only water and/or carbon dioxide as the by-product. Therefore, the reaction has high practical atom economy. In addition, this strategy could be scaled up to the gram level, and the natural product Cephamandole A could be synthesized on a mass scale.

Engineering of catalytic hairpin-rigidified Y-shaped DNA-functionalized nanomachine to rapidly detect mRNA.

The catalytic hairpin-rigidified Y-shaped DNA through layer-by-layer assembly has been fixed on the surface of copper sulfide nanoparticles for the detection of survivin mRNA. The distance between the CHA probes fixed on the Y-shaped DNA is significantly shortened. The results show that the fluorescence of this nanomachine reached the maximum value in 50 min (excitation wavelength at 488 nm and emission wavelength 526 nm), and its reaction rate is more than 5-fold faster than that of the free-CHA control system. In addition, the nanomachine showed high sensitivity (LOD of 3.5 pM) and high specificity for the survivin mRNA detection. Given its fast response time and excellent detection performance, we envision that the catalytic hairpin-rigidified Y-shaped DNA-functionalized nanomachine will offer potential applications in disease diagnostics and clinical applications.

Construction of an Ultrasensitive Molecularly Imprinted Virus Sensor Based on an "Explosive" Secondary Amplification Strategy for the Visual Detection of Viruses.

Viral outbreaks have caused great disruptions to the economy and public health in recent years. The accurate detection of viruses is a key factor in controlling and overcoming epidemics. In this study, an ultrasensitive molecularly imprinted virus sensor was developed based on an "explosive" secondary amplification strategy. Magnetic particles coated with carbon quantum dots (Fe3O4@CDs) were used as carriers and fluorescent probes, while aptamers were introduced into the imprinting layer to enhance the specific recognition of the target virus enterovirus 71 (EV71). When EV71 was captured by the imprinted particles, the fluorescence of the CDs was quenched, especially after binding to the aptamer-modified ZIF-8 loaded with a large amount of phenolphthalein, thereby resulting in signal amplification. Then, when adjusting the pH of the solution to 12, the decomposition of ZIF-8 released phenolphthalein, which turned the solution red, leading to the second "explosive" amplification of the signal. Therefore, the detection of EV71 with ultrasensitivity was achieved, which allows for visual detection by the naked eye in the absence of any instruments. The detection limits for fluorescence and visualization detection were 8.33 fM and 2.08 pM, respectively. In addition, a satisfactory imprinting factor of 5.4 was achieved, and the detection time only needed 20 min. It is expected that this fluorescence-colorimetric dual-mode virus molecularly imprinted sensor will show excellent prospects in epidemic prevention and rapid clinical diagnosis.

Radical Cross-Coupling Reaction Based on Hydrogen Atom Abstraction of DMF and Decarboxylation of α-Ketoacid under Electricity.

The cleavage of DMF into a dimethyl carbamoyl radical under mild electrochemical conditions was revealed for the first time. Meanwhile, an electrochemical decarboxylation of α-ketoacid occurred to produce an acyl radical. The radical cross-coupling reaction of these two electron-deficient acyl radicals was carried out with high selectivity. This discovery provides a new electrochemical way for the cracking of DMF, and a milder and safer method for its application in organic synthesis.

pH-activated DNA nanomachine for miRNA-21 imaging to accurately identify cancer cell.

MicroRNA (miRNA) imaging has been employed to distinguish cancer cells from normal cells by exploiting the overexpression of miRNA in cancer. Inspired by the acidic extracellular tumor microenvironment, we designed a pH-activated DNA nanomachine to enable the specific detection of cancer cells using miRNA imaging. The DNA nanomachine was engineered by assembling two hairpins (Y1 and Y2) onto the surface of a ZIF-8 metal-organic framework (MOF), which decomposed under acidic conditions to release the adsorbed DNA hairpin molecules in situ. The released hairpins were captured by the target miRNA-21 and underwent catalytic hairpin assembly amplification between Y1 and Y2. The detection limit for miRNA assays using the DNA nanomachine was determined to be 27 pM, which is low enough for sensitive detection in living cells. Living cell imaging of miRNA-21 further corroborated the application of the DNA nanomachine in the identification of cancer cell.

A sandwich sensor based on imprinted polymers and aptamers for highly specific double recognition of viruses.

Highly selective and highly efficient identification of large viruses has been a major obstacle in the field of virus detection. In this work, a novel sandwich resonance light scattering sensor was designed based on molecularly imprinted polymers (MIPs) and aptamers for the first time. One of the recognition probes was obtained by molecular imprinting using environmentally friendly carbon spheres as carriers and the other by modification of the aptamer that can specifically recognize hepatitis B virus (HBV) on the surface of silicon spheres. In the presence of both probes, an MIP-HBV-aptamer sandwich structure was formed continuously in the system with the increase in HBV concentration, resulting in a strong resonance light scattering response. Finally, satisfactory selectivity and sensitivity were obtained, and the imprinting factor was as high as 7.56, which was higher than that reported in previous works of viral molecular imprinting sensor. In addition, it is of great significance to solve the problem of insufficient selectivity of traditional detection methods for macromolecular targets.

An enzyme-free probe based on G-triplex assisted by silver nanocluster pairs for sensitive detection of microRNA-21.

A sensitive ratiometric fluorescence probe based on hybridization chain reaction (HCR) was constructed for sensitive detection of miRNA-21 by using G-triplex and silver nanocluster pairs (AgNC pairs) as an enzyme-free and label-free signal output group. miRNA-21 was used as the primer for the hybridization chain reaction of molecular beacon 1 (MB1) containing the locked G-triplex sequence and molecular beacon 2 (MB2) with intact AgNC pairs at the 5' and 3' end activation. The double-stranded product was obtained along with the opening of the G-triplex and the separation of the AgNC pairs. A detection limit of 67 pM and a linear detection range of 0.1-300 nM were obtained for miRNA-21 determination. The proposed strategy enabled the monitoring of miRNA-21 levels in at least three cell lines, indicating that it provided new ideas for detecting miRNA in real samples. MB1 and MB2 contained the locked G-triplex sequence and silver nanocluster pairs (AgNC pairs), respectively. In the presence of target, the hybridization chain reaction (HCR) between MB1 and MB2 was initiated. At the same time, the locked G-triplex was released and combined to the dye thioflavin T (THT) to increase fluorescence, while the separation of the AgNC pairs caused the fluorescence to decrease. The double-stranded (ds) DNA product was generated to form a ratiometric signal to be detected.

Specific determination of HBV using a viral aptamer molecular imprinting polymer sensor based on ratiometric metal organic framework.

An approach is reported based on the combination of aptamer and metal organic frameworks (MOF) to prepare a molecularly imprinted sensor that recognizes viruses with high specificity and sensitivity. Using MIL-101-NH2 as a polymer carrier, viral aptamers were introduced into the carrier surface through an amide reaction to specifically identify the target, and surface imprinting is carried out through tetraethyl silicate (TEOS) self-polymerization. The MIL-101-NH2 is also used as the reference fluorescence signal (λex/λem = 290/460 nm) and rhodamine B as the change signal (λex/λem = 550/570 nm). The ratiometric fluorescence detection and dual recognition strategy not only reduce environmental interference but also greatly improve the sensor's anti-interference ability, the obtained imprinting factor was 5.72, and the detection limit as low as 1.8 pmol L-1. Therefore, the molecular imprinting sensor designed realizes the specific and highly sensitive identification of viruses, which provides theoretical support for the application of molecular imprinting technology in clinical diagnosis of viruses. Graphical abstract Aptamer-molecular imprinting polymer based on metal-organic framework ratiometric fluorescent detect virus.

A novel Wulff-type boronate acid-functionalized magnetic metal-organic framework imprinted polymer for specific recognition of glycoproteins under physiological pH.

Boronate affinity molecularly imprinted materials have been widely used for the separation of glycoproteins under alkaline conditions that is not conducive to the structural stability of the protein. In this work, a kind of novel molecularly imprinted polymer (MIP/TBA/MOF@Fe3 O4 ) was prepared via grafting self-assembled molecular team of boronic acids on the surface of the magnetic metal-organic framework core. The teamed boronate affinity was formed by 2-mercaptoethylamine and 4-mercaptophenylboronic acid for specific separation of glycoproteins under physiological pH (pH 7.4). The obtained nanoparticles show high binding capacities (337.8 mg/g), fast adsorption equilibrium time (20 min), and good specificity (imprinting factor, 4.52) for glycoproteins under physiological pH. Furthermore, the prepared imprinted polymer still shows good adsorption capacity for glycoprotein after five times of repeated use, and its adsorption capacity only dropped by 4.7%. More importantly, the prepared nanoparticles have good potential to adsorb glycoproteins from real biological samples.

Single-excited double-emission CdTe@CdS quantum dots for use in a fluorometric hybridization assay for multiple tumor-related microRNAs.

A method is described for the simultaneous determination of hepatocellular carcinoma-associated microRNA-122 and microRNA-199a/b-3p. This probe consists of two kinds of nanomaterials. The first comprises CdTe@CdS core-shell quantum dots which, on excitation at 375 nm give two emissions, with peak wavelengths at 543 (g-QDs) and at 627 nm (r-QDs). The second comprises gold nanoparticles acting as a quencher. In the absence of the target, g-QD-N1 and r-QD-N2 are stable due to the fluorescence stability. With the addition of microRNA-122 and microRNA-199a/b-3p, g-QD-N1 and r-QD-N2 are conjugated to the surface of AuNP-S1/S2 through base complementary pairing. As a result, fluorescence resonance energy transfer (FRET) occurs, resulting in a decrease at 550 nm and 635 nm respectively, which can realize the simultaneous detection of two different microRNAs. Detection is achieved within 50 min. The detection limits (3σ/k) are 0.2 nM for microRNA-122 and 0.5 nM for microRNA-199a/b-3p. The clinical applicability of the assay was demonstrated by detecting microRNAs in human serum and different cell lysates. Graphical abstractSchematic for the simultaneous determination of microRNA-122 and microRNA-199a/b-3p by FRET.

Molecular imprinting resonance light scattering nanoprobes based on pH-responsive metal-organic framework for determination of hepatitis A virus.

Molecularly imprinted polymer (HM@MIP) nanoprobes were designed form the pH-responsive polymer (dimethylaminoethyl methacrylate (DMA)) and MIL-101. This probe was applied to the selective determination of hepatitis A virus (HAV) through Resonance light scattering (RLS) technique. DMA adjusts pH of the system to facilitate the capture and release of virus by HM@MIPs as anticipated. And it results in the enhancement or weaken of RLS intensity. According to RLS intensity at 470 nm, a linear concentration of 0.02-2.0 nmol·L-1 and a limit of detection of 0.1 pmol·L-1 were obtained within 20 min. The excellent recoveries ranges from 88% to 107%, and it indicates the prominent ability of the HM@MIPs to determination HAV in human serum and their potential ability to determination virus in real applications. Graphical abstractPrinciple of preparation of the HM@MIPs and detection of virus.

Visual Simultaneous Detection of Hepatitis A and B Viruses Based on a Multifunctional Molecularly Imprinted Fluorescence Sensor.

Simultaneous detection of large viruses has been a great obstacle in the field of molecular imprinting. In this work, for the first time, a multifunctional molecularly imprinted sensor for single or simultaneous determination of hepatitis A virus (HAV) and hepatitis B virus (HBV) is provided. Visual detection was realized due to the color of green and red quantum dots that varied with the concentration of the target substance. The combination of hydrophilic monomers and metal chelation reduced the nonspecific binding and enhanced the specificity of adsorption. As a result, satisfactory selectivity and sensitivity were obtained for the detection of the two viruses, with imprinting factors of 3.70 and 3.35 for HAV and HBV, and limits of detection of 3.4 and 5.3 pmol/L, respectively, that were achieved within 20 min. The excellent recoveries during simultaneous detection and single detection modes indicate the prominent ability of the proposed sensor to detect HAV and HBV in human serum and the potential ability to simultaneously detect multiple viruses in real applications.

A rapid label- and enzyme-free G-quadruplex-based fluorescence strategy for highly-sensitive detection of HIV DNA.

Because rapid, convenient, and selective methods for HIV detection are urgently needed, herein, a simple label-free and enzyme-free strategy is constructed for sensitive fluorescence detection of HIV DNA using the fluorescent intercalating dye thioflavin T (THT) as the detection signal source. This strategy utilizes a hairpin DNA sequence (H1) and two assistant strands. H1 is wisely designed with a G-quadruplex sequence in the stem. Target DNA, when present in solution, will hybridize with H1 to form H1/target duplexes and release the G-quadruplexes. Additionally, the assistant probes hybridize with the unfolded H1 to form a stable DNA double strand, resulting in the displacement of the target to participate in another similar reaction cycle. Consequently, many G-quadruplex structures are generated, leading to a significantly amplified fluorescence signal of THT. The linear range is from 0.1 nM to 50.0 nM with a limit of detection of 13 pM. Results can be achieved within 40 min, because the cyclic amplification involves only one DNA hairpin and two auxiliary chains. Furthermore, this platform exhibited good selectivity with one base mismatch or other DNA sequences. This strategy could be used as a simple, sensitive, and selective tool to detect other DNA biomarkers.

DNA-programming multicolor silver nanoclusters for sensitively simultaneous detection of two HIV DNAs.

A novel DNA-stabilized silver nanoclusters (AgNCs)-based label-free fluorescent platform for simultaneously detecting two human immunodeficiency virus oligonucleotides (HIV DNAs) was developed. The sensing platform was established based on fluorescence enhancement of guanine (G)-rich and the phenomenon in the process of two silver nanoclusters (AgNCs) forming a nanoclusters dimer. The probe (AgNCs/G) utilized for HIV-1 detection adopted an effective conformation based on enhancement effect of G-rich sequence (at 500 nm ex / 565 nm em) while the probe (AgNCs/AgNCs) for HIV-2 generated fluorescence signals (at 580 nm ex / 630 nm em) with bright fluorescence only in nanoclusters dimer. The nanoprobe shows high selectivity for multiplexed analysis of target DNA with a detection limit of 11 pM, respectively. Moreover, this is the first time to use the affectivity of fluorescent AgNCs and two HIV DNAs simultaneous detection integrated into a novel method, which shows a great promise in biomedical research and early clinical diagnosis.

A general scheme for fluorometric detection of multiple oligonucleotides by using RNA-cleaving DNAzymes: application to the determination of microRNA-141 and H5N1 virus DNA.

A widely applicably method is described for fluorometric determination of targets such as microRNA and viral DNA. It is making use of a Mg(II)-dependent DNAzyme and a G-quadruplex. In the absence of analyte, an inactive DNAzyme is formed by the hybridization of split DNAzymes and substrate. On addition of target analyte, the end of each strand of the split DNAzymes bind the analyte. This leads to the generation of an active DNAzyme. In the presence of Mg(II), the activated DNAzyme is formed and can cleave the substrate strand. Hence, the caged G-quadruplex sequences will be released. These released G-quadruplexes combine with thioflavin T to generate a G-quadruplex/thioflavin T complex and thereby cause amplified fluorescence. The method shows a 70 pM detection limit for H5N1 and works over a wide linear range 1 nM to 400 nM. Conceivably, this detection scheme has a wide scope in that it may be applied to other assays for microRNAs and DNAs by variation of the type of DNAzyme. Graphical abstract Schematic presentation of target detection: the DNAzyme cannot cleave the substrate strand when target is absent. Once the target is added, the active DNAzyme can cleave the substrate strand in the presence of Mg2+, resulting in significant fluorescence enhancement when the release of the caged G-quadruplex sequences binding with 2-[4-(dimethylamino)phenyl]-3,6-dimethylbenzothiazolium chloride (ThT).

An ultrasensitive guanine wire-based resonance light scattering method using G-quadruplex self-assembly for determination of microRNA-122.

An enzyme-free resonance light scattering (RLS) method is described for the determination of microRNA-122. A guanine nanowire (G-wire) is used that consists of a predesigned DNA1 and a G-quadruplex sequence DNA2. These hybridize with microRNA-122 and partially hybridize with DNA2. After formation of stable double strands with DNA1, DNA2 is released. On addition of K+ and Mg2+ ions, the G-quadruplex sequences undergo self-assembly to form long filamentous G-wires. This increases the intensity of RLS. A 6.1 pM detection limit was obtained, and the linear response covers the 50 pM to 300 nM microRNA concentration range. The method was successfully applied to the quantitation of microRNA-122 in hepatocellular carcinoma cell lysates. Conceivably, this assay can be extended to other RLS methods for biomarker detection by simply changing the sequence of DNA1. Graphical abstract The G-quadruplex sequences of DNA2 were locked with DNA1. The G-quadruplex fragments of DNA2 were released after the hybridization of microRNA-122 with DNA1. These liberated G-quadruplex sequences were self-assembled into long filamentous guanine nanowires (G-wires) which increased resonance light intensity in the presence of Mg2+.

Double G-quadruplexes in a copper nanoparticle based fluorescent probe for determination of HIV genes.

A DNA-templated copper nanoparticle (CuNP) probe has been developed for the determination of the human immunodeficiency virus oligonucleotide (HIV-DNA). The function of the probe relies on affinity binding-induced DNA hybridization associated with the use of double G-quadruplexes. Double-stranded DNA (dsDNA) with poly(AT-TA) bases was used as a template for synthesis of dsDNA-CuNPs. These have weak fluorescence. In the next step, two G-rich sequences that are linked to both sides of the ds-DNA are locked by HIV complementary DNA (cDNA). If HIV-DNA is introduced, it will hybridize with cDNA, thereby transforming the two G-rich sequences into G-quadruplexes. This enhances the fluorescence of the adjacent dsDNA-CuNPs. Fluorescence increases linearly in the 1 to 200 and 250-1000 nM HIV-DNA concentration range, and the detection limit is 13 pM. This enzyme-free fluorometric assay is time-saving, easily operated, and therefore has large potential in biosensing because it may be extended to various other DNA targets. Graphic abstract Double-strand DNA-templated copper nanoparticles (DNA-CuNPs) have weak fluorescence. When Human Immunodeficiency Virus oligonucleotide (HIV-DNA) is added, it completely hybridized with HIV complementary DNA (cDNA). As a result, the two exposed G-rich sequences are transformed into G-quadruplexes, and an apparent increase in the fluorescence intensity can be observed. (AA: ascorbic acid).

Surface-imprinted microspheres prepared by a template-oriented method for the chiral separation of amlodipine.

The surface imprinting technique has been developed to overcome the mass-transfer difficulty, but the utilization ratio of template molecules in the imprinting procedure still remains a challengeable task to be improved. In this work, specifically designed surface-imprinted microspheres were prepared by a template-oriented method for enantioseparation of amlodipine besylate. Submicron mesoporous silica microspheres were surface-modified with double bonds, followed by polymerizing methacrylic acid to generate carboxyl modified mesoporous silica microspheres (PMAA@SiO2 ). Afterwards, PMAA@SiO2 was densely adsorbed with (S)-amlodipine molecules to immobilize template molecules through multiple hydrogen bonding interactions. Then surface molecular imprinting was carried out by cross-linking the carboxyl group of PMAA@SiO2 with ethylene glycol diglycidyl ether. The surface-imprinted microspheres showed fast binding kinetics of only 20 min for equilibrium adsorption, and the saturation adsorption capacity reached 137 mg/g. The imprinted materials displayed appreciable chiral separation ability when used as column chromatography for enantioseparation of amlodipine from amlodipine besylate, and the enantiomeric excess of (S)-amlodipine reached 13.8% with only 2.3 cm column length by no extra chiral additives. Besides, the imprinted materials exhibited excellent reusability, and this allows the potential application for amplification production of amlodipine enantiomer.