Sex differences in fetal brain functional network topology.

The fetal period is a critical stage in brain development, and understanding the characteristics of the fetal brain is crucial. Although some studies have explored aspects of fetal brain functional networks, few have specifically focused on sex differences in brain network characteristics. We adopted the graph theory method to calculate brain network functional connectivity and topology properties (including global and nodal properties), and further compared the differences in these parameters between male and female fetuses. We found that male fetuses showed an increased clustering coefficient and local efficiency than female fetuses, but no significant group differences concerning other graph parameters and the functional connectivity matrix. Our study suggests the existence of sex-related distinctions in the topological properties of the brain network at the fetal stage of development and demonstrates an increase in brain network separation in male fetuses compared with female fetuses.

A ratiometric fluorescence assay for the detection of DNA methylation based on an alkaline phosphatase triggered in situ fluorogenic reaction.

The accurate and sensitive quantification of DNA methylation is significant for the early diagnosis of cancer. In this work, an alkaline phosphatase (ALP) triggered in situ fluorogenic reaction between ascorbic acid (AA) and 2,3-DAN was employed as a ratiometric fluorescent probe for the accurate and sensitive detection of DNA methylation with the assistance of ALP encapsulated liposomes. The quinoxaline derivative with a yellow fluorescence emission (I525) was generated from the reaction between AA and 2,3-DAN. Meanwhile, the consumption of 2,3-DAN declined its fluorescence intensity (I386). A ratiometric fluorescent probe (I525/I386) constructed by the above in situ fluorogenic reaction was applied for the accurate detection of DNA methylation. The methylated DNA was first captured by its complementary DNA in 96-well plates. Then, 5mC antibody (Ab) linked liposomes that were encapsulated with ALP recognized and combined with the methylation sites of the target DNA. After the liposomes were lysed by Triton X-100, the released ALP triggered the hydrolysis of ascorbic acid diphosphate (AAP) to form AA, participating in the fluorogenic reaction with 2,3-DAN to produce a quinoxaline derivative. Thus, the ratiometric fluorescence detection of DNA methylation was achieved using I525/I386 values. Using the ALP-enzyme catalyzed reaction and liposomes as signal amplifiers, a low detection limit of 82 fM was obtained for DNA methylation detection. Moreover, the accuracy of the assay could be improved using ratiometric fluorescent probes. We hope that the proposed assay will pave a new way for the accurate determination of low-abundance biomarkers.

ALP-assisted chemical redox cycling signal amplification for ultrasensitive fluorescence detection of DNA methylation.

Affinity assays allow direct detection of DNA methylation events without requiring a special sequence. However, the signal amplification of these methods heavily depends on nanocatalysts and bioenzymes, making them suffer from low sensitivity. In this work, alkaline phosphatase (ALP)-assisted chemical redox cycling was employed to amplify the sensitivity of fluorescence affinity assays for DNA methylation detection using Ru@SiO2@MnO2 nanocomposites as fluorescent probes. In the ALP-assisted chemical redox cycling reaction system, ALP hydrolyzed 2-phosphate-L-ascorbic acid trisodium salt (AAP) to produce AA, which could reduce MnO2 nanosheets to form Mn2+, making the fluorescence recovery of Ru@SiO2 nanoparticles possible. Meanwhile, AA was oxidized to dehydroascorbic acid (DHA), which was re-reduced by tris(2-carboxyethyl) phosphine (TCEP) to trigger a redox cycling reaction. The constantly generated AA could etch large amounts of MnO2 nanosheets and greatly recover Ru@SiO2 fluorescence, amplifying the signal of the fluorescence assay. Employing the proposed ALP-assisted chemical redox cycling signal amplification strategy, a sensitive affinity assay for DNA methylation detection was achieved using ALP encapsulated liposomes that were linked with the 5mC antibody (Ab) to bind with methylated sites. A detection limit down to 2.9 fM was obtained for DNA methylation detection and a DNA methylation level as low as 0.1% could be distinguished, which was superior to conventional affinity assays. Moreover, the affinity assays could detect DNA methylation more specifically and directly, implying their great potential for the analysis of tumor-specific genes in liquid biopsy.

A Fluorescence Turn On-off-on Method for Sensitive Detection of Sn2+ and Glycine Using Waste Eggshell Membrane Derived Carbon Nanodots as Probe.

Changes in Sn2+ and glycine levels are relevant to many important physiological procedures in human health. However, investigation of their physiological functions is limited because few versatile methods towards Sn2+ and glycine detection have been developed. In this work, a fluorescence turn on-off-on strategy was firstly constructed for rapid and sensitive detection of Sn2+ and glycine through the specific binding between Sn2+ and glycine. Carbon nanodots (CDs) with a quantum yield of 19.5% were synthesized by utilizing inner film of waste eggshell as carbon source and employed as fluorescent probe. In the presence of Sn2+, the fluorescence of CDs was quenched by Sn2+ via the primary inner filter effect (IFE). However, the binding between Sn2+ and glycine prevented the IFE between Sn2+ and CDs, resulting in fluorescence recovery of CDs. Under optimized conditions, the fluorescent response of CDs displayed good linear relationships with the concentrations of Sn2+ in the range of 10-200 µM and 200-5000 µM, and the limit of detection (LOD) was 2.4 µM. For glycine detection, a good linear relationship was obtained in the concentration range of 5-1000 µM with a low LOD down to 0.76 µM. Moreover, the practicability of the assay was also demonstrated by measuring glycine content in human serum samples. This work provides an economical, green and fast method for biological analysis of Sn2+ and glycine.

Single Biomolecule Imaging by Electrochemiluminescence.

Herein, a single biomolecule is imaged by electrochemiluminescence (ECL) using Ru(bpy)32+-doped silica/Au nanoparticles (RuDSNs/AuNPs) as the ECL nanoemitters. The ECL emission is confined to the local surface of RuDSNs leading to a significant enhancement in the intensity. To prove the concept, a single protein molecule at the electrode is initially visualized using the as-prepared RuDSN/AuNPs nanoemitters. Furthermore, the nanoemitter-labeled antibody is linked at the cellular membrane to image a single membrane protein at one cell, without the interference of current and optical background. The success in single-biomolecule ECL imaging solves the long-lasting task in the ultrasensitive ECL analysis, which should be able to provide more elegant information about the protein in cellular biology.

Turning waste into treasure: chicken eggshell membrane derived fluorescent carbon nanodots for the rapid and sensitive detection of Hg2+ and glutathione.

Herein, a green, economical, and waste-utilization approach is reported for the synthesis of water-soluble carbon nanodots (C-Dots) with a high fluorescence quantum yield of 19.5%. As a common protein-rich waste, eggshell membrane was selected as a cost-effective and ideal precursor to prepare C-Dots using the microwave method. The as-prepared C-Dots showed a maximum emission at 375 nm with an excitation wavelength at 235 nm. The fluorescent C-Dots were adopted as a sensitive probe for the rapid detection of Hg2+ and glutathione (GSH) based on the fluorescence off and on (turn-off-on) strategy. This was ascribed to the fact that Hg2+ could effectively quench the fluorescence of the C-Dots and GSH was able to prevent fluorescence quenching owing to the specific binding between Hg2+ and GSH. The designed method exhibited a high sensitivity and selectivity towards the detection of Hg2+ and GSH. Under the optimized conditions, the method showed a good linear relationship with Hg2+ concentration in the range from 100 nM to 50 µM with a detection limit of 32.0 nM. For GSH detection, it displayed a linear range from 50 nM to 10 µM with a detection limit of 9.8 nM. Moreover, this method was successfully applied to detect GSH in human serum samples. The eggshell derived fluorescent C-Dots pave the way for economical environmental and biological analyses.

L-Histidine-DNA interaction: a strategy for the improvement of the fluorescence signal of poly(adenine) DNA-templated gold nanoclusters.

An interesting phenomenon is described that the fluorescence signal of poly(adenine) (A) DNA-templated gold nanoclusters (AuNCs) is greatly improved in the presence of L-histidine by means of L-histidine-DNA interaction. The modified nanoclusters display strong fluorescence emission with excitation/emission maxima at 290/475 nm. The fluorescence quantum yield (QY) is improved from 1.9 to 6.5%. Fluorescence enhancement is mainly ascribed to the L-histidine-DNA interaction leading to conformational changes of the poly(A) DNA template, which offer a better microenvironment to protect AuNCs. The assay enables L-histidine to be determined with good sensitivity and a linear response that covers the 1 to 50 nM L-histidine concentration range with a 0.3 nM limit of detection. The proposed method has been applied to the determination of imidazole-containing drugs in pharmaceutical samples. A turn-on fluorescent method has been designed for the sensitive detection of L-histidine as well as imidazole-containing drugs on the basis of the L-histidine-DNA interaction.

Single-Molecule Fluorescence Imaging for Ultrasensitive DNA Methyltransferase Activity Measurement and Inhibitor Screening.

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single-molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently, the streptavidin-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001-1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Three-Dimensional Plasmonic Trap Array for Ultrasensitive Surface-Enhanced Raman Scattering Analysis of Single Cells.

Single-cell analysis provides an important strategy to evaluate cellular heterogeneity. Although surface-enhanced Raman scattering (SERS) has been considered as a promising label-free technique for single-cell analysis, it remains at the early stage for characterizing the extracellular metabolites of single cells. Herein, we developed a convenient, flexible, and straightforward three-dimensional (3D) plasmonic trap array for simultaneously compartmentalizing and sensitively detecting single-cell metabolites. The 3D trap was spontaneously self-formed by an interfacial-energy-driven process when a liquid droplet was covered with an immiscible oil liquid (polydimethylsiloxane, PDMS). When a droplet of pure AgNO3 solution was immersed into PDMS, Ag+ ions were automatically reduced by the residual Si-H groups in PDMS. Snowflake-like nanoparticles of Ag could be formed on the inner surface of the 3D traps by tuning the concentration of Ag salt precursors and then assembled to flower-like microstructures, endowing the traps with remarkable plasmon enhancement. The established 3D traps exhibited considerably enhanced surface plasmon resonance signals for Raman reporting, and a low detection limit at the aM level was achieved for p-aminothiophenol. Moreover, these 3D traps can serve as an efficient tool for single-cell SERS measurement. As a proof-of-concept, dipicolinic acid, a common biomarker of bacterial spores, was successfully detected from a single cell. The presented approach provides a versatile tool for label-free and sensitive detection of single-cell environments.

Single Molecule Fluorescent Colocalization of Split Aptamers for Ultrasensitive Detection of Biomolecules.

Single-molecule fluorescence imaging is a promising strategy for biomolecule detection. However, the accuracy of single-molecule method is often compromised by the false-positive events at the ultralow sample levels that are caused by the nonspecific adsorption of the fluorescent labeled probe and other fluorescent impurities on the imaging surface. Here, we demonstrate an ultrasensitive single molecule detection assay based on dual-color fluorescent colocalization of spilt aptamers that was implemented to the measurement of adenosine triphosphate (ATP). The ATP aptamer was split into two fragments and labeled with green and red dye molecules, respectively. When the two probes of split aptamers were brought together by the target ATP molecule, the two colors of fluorescence of two probes were simultaneously detected through two channels and projected to the correlated locations in the two halves of image. The colocalizaiton imaging of two split apatamer probes greatly excluded the false detection of biomolecules that was usually caused by the fluorescent noise of single nonbound aptamer probes and impurities, and further improved the accuracy of measurement. The assay showed excellent selectivity and high sensitivity for ATP detection with linear range of 1 pM to 5 nM and a detection limit of 100 fM. This versatile protocol of single molecule colocalization of split apatamer can be widely applied to the ultrasensitive and highly accurate detection of many types of biomolecules in basic research and biomedical applications.

Tannic acid-accelerated fenton chemical reaction amplification for fluorescent biosensing: The proof-of-concept towards ultrasensitive detection of DNA methylation.

As a promising biomarker, the level of methylated DNA usually changes in the early stage of the cancer. Ultrasensitive detection of the changes of methylated DNA offers possibility for early diagnosis of cancer. In this work, a tannic acid-accelerated Fenton chemical reaction amplification was firstly proposed for the construction of ultrasensitive fluorescent assay. Tannic acid was used as reductant to accelerate Fenton reaction procedure through the conversion of Fe3+/Fe2+, generating hydroxyl radicals (·OH) continuously. The produced ·OH oxidized massive non-fluorescent terephthalic acid (TA) to fluorescent-emitting hydroxy terephthalic acid (TAOH). In this way, the fluorescent signal could be greatly enhanced and the sensitivity was improved almost 116 times. The proposed signal amplification strategy was further applied to detect of DNA methylation with the assistance of liposome encapsulated with tannic-Fe3+ complexes. The methylated DNA was firstly captured through the hybridization with its complementary DNA that were pre-modified in the 96-well plate via the combination between streptavidin (SA) and biotin. Then, 5 mC antibody on the surface of liposomes specially recognized and combined with methylation sites, which brought large amount of tannic-Fe3+ complexes to participate Fenton reaction. The fluorescence of generated TAOH was depended on the concentration of methylated DNA. The assay showed good analytical performance for methylated DNA with a limit of detection (LOD) of 1.4 fM. It's believed that tannic acid-accelerated Fenton chemical reaction amplification strategy provides a promising platform for ultrasensitive fluorescent detection of low abundant biomarkers.

Glutathione-stabilized copper nanoclusters mediated-inner filter effect for sensitive and selective determination of p-nitrophenol and alkaline phosphatase activity.

A simple and highly selective fluorescence biosensor has been exploited for p-nitrophenol (p-NP) and alkaline phosphatase (ALP) activity detection based on the glutathione-stabilized copper nanoclusters (GSH-CuNCs) mediated-inner filter effect (IFE). The GSH-CuNCs were prepared by employing GSH as stabilizer and ascorbic acid (AA) as reductant. The obtained GSH-CuNCs exhibited a strong blue fluorescence emission at 420 nm with an excitation wavelength of 365 nm, which overlapped largely with the absorption spectra of p-nitrophenol (p-NP). Therefore, the luminescence of GSH-CuNCs could be quenched by p-NP through inner filter effect. In addition, ALP catalyzed the substrate p-nitrophenyl phosphate (p-NPP) to form p-nitrophenol (p-NP), which also leading to the fluorescence quenching of GSH-CuNCs. The fluorescent strategy was realized for the sensitive determination of p-NP and ALP activity with the promising limit of detection of 20 nM (for p-NP) and 0.003 mU⋅mL-1 (for ALP). Furthermore, the method could be applied to detect the p-NP content in river water samples and ALP activity in human serum samples.

In-situ formation of MnO2 nanoparticles on Ru@SiO2 nanospheres as a fluorescent probe for sensitive and rapid detection of glutathione.

Glutathione (GSH)-switched fluorescent assays have appealed much attention due to rapid signal changes of fluorescent probes. However, exposure to exterior environment of fluorescent probe causes photobleaching and premature leakage, leading to low sensitivity and poor photostability. Herein, luminescent SiO2 nanoparticles encapsulated with Ru(bpy)32+ (Ru@SiO2) were designed and synthesized as fluorescent probe to construct a GSH-switched fluorescent assay. The encapsulation of Ru(bpy)32+ in the SiO2 nanoparticles could effectively prevent the leakage of Ru(bpy)32+ molecules, improving the photostability of probe. The fluorescence of Ru@SiO2 nanoparticles was quenched by coating MnO2 nanoparticles on Ru@SiO2 surface (Ru@SiO2@MnO2 nanocomposites) through an in situ growth approach, which reduced background of the assay. The MnO2 nanoparticles not only further inhibited the leakage of Ru(bpy)32+ molecules, but also could serve as a recognition unit of GSH. In the presence of GSH, the MnO2 nanoparticles on the surface of Ru@SiO2 nanoparticles were reduced to Mn2+, resulting the fluorescence recovery of Ru@SiO2 nanoparticles. Thus, a signal-on fluorescent strategy was constructed for GSH detection. The assay displayed good analytical performance for GSH detection with a low detection limit of 16.2 nM due to excellent fluorescence quenching ability of MnO2 nanoparticles and special role of Ru@SiO2 nanoparticles to block probe leakage. The proposed assay was also applied to measure GSH levels in human serum samples. This work paves a new way to detect GSH with high sensitivity.

Liposome-assisted chemical redox cycling strategy for advanced signal amplification: A proof-of-concept toward sensitive electrochemiluminescence immunoassay.

This work presents a novel signal amplification strategy for electrochemiluminescence (ECL) biosensor based on liposome-assisted chemical redox cycling for in situ formation of Au nanoparticles (Au NPs) on TiO2 nanotubes (TiO2 NTs) electrode. The system was exemplified by ascorbic acid (AA)-loaded liposome, the redox cycling of AA utilizing tris (2-carboxyethyl) phosphine (TCEP) as reductant, and the use of Au nanoclusters (Au NCs)/TiO2 NTs as working electrode to implement the ECL detection of prostate specific antigen (PSA). Specifically, the AA-loaded liposomes were used as tags to label the captured PSA through a sandwich immunoreaction. After the lysate of the liposome was transferred onto the interface of Au NCs/TiO2 NTs in the presence of Au3+ and TECP, the chemical redox cycling was triggered. In the cycling, Au3+ was directly reduced in situ by AA to form Au NPs on Au NCs/TiO2 NTs electrode, whereas the oxidation product of AA was reduced by TCEP to regenerate AA. The large loading capacity of the liposome and chemical redox cycling resulted in the incomplete reduction of the Au NCs to Au NPs on the TiO2 NTs electrode, enhancing the ECL intensity greatly. The multiple signal amplification strategy achieved an ultrasensitive detection for PSA with a detection limit down to 6.7 × 10-15 g mL-1 and a wide linear concentration range from 1.0 × 10-14 to 1.0 × 10-8 g mL-1. It is believed that this work is anticipated to extend the employment of advanced chemical redox cycling reaction in the field of ECL bioassays.

Iodide-modified Ag nanoparticles coupled with DSN-Assisted cycling amplification for label-free and ultrasensitive SERS detection of MicroRNA-21.

With the emergence of microRNA (miRNA) as a key player in early clinical disease diagnosis, development of rapidly sensitive and quantitative miRNA detection methods are imperative. Herein, a label-free SERS assay coupled with duplex-specific nuclease (DSN) signal amplification strategy was proposed for facilely ultrasensitive and quantitative analysis of miRNA-21. Firstly, magnetic beads assembled with excessive capture DNA were utilized to hybridize the target miRNA-21. These DNA-RNA heteroduplexes were cleaved by DSN to generate small nucleotide fragments into the supernatant and the miRNA-21 released and rehybridized another DNA, going to the next DSN cycle. Consequently, numerous of small nucleotide fragments of capture DNA were released from magnetic beads and the miRNA-21 signal was transferred and amplified by the SERS signals of total phosphate backbones which are abundant in nucleotide. Furthermore, iodide-modified Ag nanoparticles (AgINPs) was employed to generate a strong and reproducible SERS signal. The proposed method displayed excellent performance for miRNA-21 detection with the linear range from 0.33 fM to 3.3 pM, and a lower detection limit of 42 aM. Moreover, this strategy exhibited effectively base discrimination capability and was successfully applied for monitoring the expression levels of miRNA-21 in different cancer cell lines and human serum.

Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging.

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5'-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

A fluorescent biosensor for protein detection based on poly(thymine)-templated copper nanoparticles and terminal protection of small molecule-linked DNA.

In this paper, a fluorescent biosensor has been developed for protein detection based on poly(thymine) (poly T)-templated copper nanoparticles (Cu NPs) and terminal protection of small molecule linked-DNA. This strategy was demonstrated by using small molecule biotin and its binding protein streptavidin (SA) as a model case. In this assay, biotin-linked poly T (biotin-T30) probe was specifically bound to the target protein SA with strong affinity in the presence of SA. The selective binding events confirmed that biotin-T30 probe was protected against the hydrolysis by exonuclease I (Exo I), which could effectively template the formation of fluorescent Cu NPs. The results revealed that the developed strategy was highly sensitive for detecting SA in the concentration range from 0.5 to 1000 nM with a detection limit of 0.1 nM. In addition, the relative standard deviation was 3.6% in 5 repetitive assays of 50 nM SA, which indicated that the reproducibility of the method was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, low cost, and simplified operations. Thus, it could hold considerable potential to construct a simple, selective and sensitive fluorescent platform for detection of small molecule-protein interactions in molecular diagnostics and genomic research.