Dual-Mode Arginine Assay Based on the Conformation Switch of a Ferrocene-Grafted Polypeptide.

Both controllable regulation of the conformational structure of a polypeptide and specific recognition of an amino acid are still arduous challenges. Here, a novel dual-mode (electrochemical and colorimetric) biosensor was built for arginine (Arg) recognition based on a conformation switch, utilizing controllable and synergistic self-assembly of a ferrocene-grafted hexadecapeptide (P16Fc) with gold nanoparticles (AuNPs). Benefiting from the flexibility and unique topological structure of P16Fc formed nanospheres, the assembly and disassembly can undergo a conformation transition induced by Arg through controlling the distance and number of Fc detached from the gold surface, producing on-off electrical signals. Also, they can induce aggregation and dispersion of AuNPs in solution, causing a color change. The mechanism of Arg recognition with polypeptide conformation regulation was well explored by combining microstructure characterizations with molecular mechanics calculations. The electrochemical and colorimetric assays for Arg were successfully established in sensitive and selective manner, not only obtaining a very low detection limit, but also effectively eliminating the interference from other amino acids and overcoming the limitation of AuNP aggregation. Notably, the conformational change-based assay with the peptide regulated by the target will make a powerful tool for the amino acid biosensing and health diagnosis.

Development of surface-enhanced Raman scattering-sensing Method by combining novel Ag@Au core/shell nanoparticle-based SERS probe with hybridization chain reaction for high-sensitive detection of hepatitis C virus nucleic acid.

The ultrasensitive detection of hepatitis C virus (HCV) nucleic acid is crucial for the early diagnosis of hepatitis C. In this study, by combining Ag@Au core/shell nanoparticle (Ag@AuNP)-based surface-enhanced Raman scattering (SERS) tag with hybridization chain reaction (HCR), a novel SERS-sensing method was developed for the ultrasensitive detection of HCV nucleic acid. This SERS-sensing system comprised two different SERS tags, which were constructed by modifying Ag@AuNP with a Raman reporter molecule of 4-ethynylbezaldehyde, two different hairpin-structured HCR sequences (H1 or H2), and a detection plate prepared by immobilizing a capture DNA sequence onto the Ag@AuNP layer surface of the detection wells. When the target nucleic acid was present, the two SERS tags were captured on the surface of the Ag@AuNP-coated detection well to generate many "hot spots" through HCR, forming a strong SERS signal and realizing the ultrasensitive detection of the target HCV nucleic acid. The limit of detection of the SERS-sensing method for HCV nucleic acid was 0.47 fM, and the linear range was from 1 to 105 fM.

Deep-Reinforcement-Learning-Based Joint Energy Replenishment and Data Collection Scheme for WRSN.

With the emergence of wireless rechargeable sensor networks (WRSNs), the possibility of wirelessly recharging nodes using mobile charging vehicles (MCVs) has become a reality. However, existing approaches overlook the effective integration of node energy replenishment and mobile data collection processes. In this paper, we propose a joint energy replenishment and data collection scheme (D-JERDG) for WRSNs based on deep reinforcement learning. By capitalizing on the high mobility of unmanned aerial vehicles (UAVs), D-JERDG enables continuous visits to the cluster head nodes in each cluster, facilitating data collection and range-based charging. First, D-JERDG utilizes the K-means algorithm to partition the network into multiple clusters, and a cluster head selection algorithm is proposed based on an improved dynamic routing protocol, which elects cluster head nodes based on the remaining energy and geographical location of the cluster member nodes. Afterward, the simulated annealing (SA) algorithm determines the shortest flight path. Subsequently, the DRL model multiobjective deep deterministic policy gradient (MODDPG) is employed to control and optimize the UAV instantaneous heading and speed, effectively planning UAV hover points. By redesigning the reward function, joint optimization of multiple objectives such as node death rate, UAV throughput, and average flight energy consumption is achieved. Extensive simulation results show that the proposed D-JERDG achieves joint optimization of multiple objectives and exhibits significant advantages over the baseline in terms of throughput, time utilization, and charging cost, among other indicators.

Endogenous Enzyme-Activatable Spherical Nucleic Acids for Spatiotemporally Controlled Signal Amplification Molecular Imaging and Combinational Tumor Therapy.

Due to the adjustable hybridization activity, antinuclease digestion stability, and superior endocytosis, spherical nucleic acids (SNAs) have been actively developed as probes for molecular imaging and the development of noninvasive diagnosis and image-guided surgery. However, since highly expressed biomarkers in tumors are not negligible in normal tissues, an inevitable background signal and the inability to precisely release probes at the chosen region remain a challenge for SNAs. Herein, we proposed a rationally designed, endogenous enzyme-activatable functional SNA (Ep-SNA) for spatiotemporally controlled signal amplification molecular imaging and combinational tumor therapy. The self-assembled amphiphilic polymer micelles (SM-ASO), which were obtained by a simple and rapid copper-free strain-promoted azide-alkyne cycloaddition click reaction between dibenzocyclooctyne-modified antisense oligonucleotide and azide-containing aliphatic polymer polylactic acid, were introduced as the core elements of Ep-SNA. This Ep-SNA was then constructed by connecting two apurinic/apyrimidinic (AP) site-containing trailing DNA hairpins, which could occur via a hybridization chain reaction in the presence of low-abundance survivin mRNA to SM-ASO through complementary base pairing. Notably, the AP site-containing trailing DNA hairpins also empowered the SNA with the feasibility of drug delivery. Once this constructed intelligent Ep-SNA nanoprobe was specifically cleaved by the highly expressed cytoplasmic human apurinic/apyrimidinic endonuclease 1 in tumor cells, three key elements (trailing DNA hairpins, antisense oligonucleotide, and doxorubicin) could be released to enable subsequent high-sensitivity survivin mRNA imaging and combinational cancer therapy (gene silencing and chemotherapy). This strategy shows great application prospects of SNAs as a precise platform for the integration of disease diagnosis and treatment and can contribute to basic biomedical research.

A New Gene SCY3 Homologous to Scygonadin Showing Antibacterial Activity and a Potential Role in the Sperm Acrosome Reaction of Scylla paramamosain.

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3. The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin, SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus, but not by Staphylococcus aureus. The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus. Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain.

Single Molecule-Level Detection via Liposome-Based Signal Amplification Mass Spectrometry Counting Assay.

Because of the low atomization and/or ionization efficiencies of many biological macromolecules, the application of mass spectrometry to the direct quantitative detection of low-abundance proteins and nucleic acids remains a significant challenge. Herein, we report mass spectrum tags (MS-tags) based upon gold nanoparticle (AuNP)-templated phosphatidylcholine phospholipid (DSPC) liposomes, which exhibit high and reliable signals via electrospray ionization (ESI). Using these MS-tags, we constructed a liposome signal amplification-based mass spectrometric (LSAMS) "digital" counting assay to enable ultrasensitive detection of target nucleic acids. The LSAMS system consists of liposomes modified with a gold nanoparticle core and surface-anchored photocleavable DNA. In the presence of target nucleic acids, the modified liposome and a magnetic bead simultaneously hybridize with the target nucleic acid. After magnetic separation and photolysis, the MS-tag is released and can be analyzed by ESI-MS. At very low target concentrations, one liposome particle corresponds to one target molecule; thus, the concentration of the target can be estimated by counting the number of liposomes. With this assay, hepatitis C (HCV) virus RNA was successfully analyzed in clinical samples.

Colorimetric nano-beacon and magnetic separation-based rapid and visual assay for gram-negative bacteria.

Food-borne diseases caused by pathogenic bacteria are one of the serious factors affecting human health. However, the most commonly used detection methods for pathogenic bacteria not only require expensive instruments, but also take a long time due to the complicated and cumbersome detection process. Therefore, the development of a fast, simple, and low-cost detection method for pathogenic bacteria is crucial for food safety and human health. In this work, based on the high binding ability of antimicrobial peptide (AMP) and polymyxin B (PMB) to bacteria, combined with magnetic separation technology, a new enzyme-free colorimetric strategy was constructed to achieve visual detection of Gram-negative bacteria in complex samples. The sensor system was divided into the following two parts: a colorimetric signal amplification nanoprobe, which was modified with AMP to enable effective binding of the colorimetric probe to the surface of bacteria, and a PMB-modified magnetic nanobead (MNB), which was used as the capture and enrichment unit of Gram-negative bacteria, as a result of which PMB could effectively distinguish Gram-negative bacteria from Gram-positive bacteria. Under optimized conditions, the detection limit of the method for Gram-negative bacteria (e.g. E. coli (G-)) was as low as 10 CFU/mL, and it was successfully applied to complex real samples. In addition, the developed colorimetric sensor offered advantages, such as fast response, less time consumption, high sensitivity, and low cost. It can be expected to become a new diagnostic tool for on-site detection of pathogenic bacteria in remote areas.

Microemulsion-Confined Biomineralization of PEGylated Ultrasmall Fe3O4 Nanocrystals for T2-T1 Switchable MRI of Tumors.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are a novel T1 contrast agent with good biocompatibility and switchable imaging signal that have not been widely applied for magnetic resonance imaging (MRI) because it is difficult to induce their relatively close ideal agglomeration. Here, by combining the microemulsion method with the biomineralization principle, a pH-responsive T2-T1 switchable MRI nanoprobe was constructed via the microemulsion-confined biomineralization of PEGylated USPIONs (PEG-USPIONs). The size of the formed CaCO3-coated PEG-USPION conjugates (PEG-USPIONs@CaCO3 nanoprobe) was uniform and controllable, and the preparation method was simple. The PEG-USPIONs inside the nanoconjugates agglomerate more tightly, and the T1-MRI signal of the nanoprobe is converted to the T2-MRI signal. When exposed to the acidic environment of the tumor tissue or internal organelles, the CaCO3-coating of the nanoprobes is dissolved, and free PEG-USPIONs are released, thus realizing the T1-weighted imaging of the tumors. The suitability of the PEG-USPIONs@CaCO3 nanoprobe for tumor MRI detection was successfully demonstrated using a mouse model bearing a subcutaneous 4T1 xenograft.

Photoactivatable Red Chemiluminescent AIEgen Probe for In Vitro/Vivo Imaging Assay of Hydrazine.

Here, we have developed a novel photoactivatable red chemiluminescent AIEgen probe (ACL), based on the combination of the red-emission AIEgen fluorophore (TPEDC) that shows excellent singlet oxygen (1O2)-generation ability and the precursor of Schaap's dioxetane (the linker connected to adamantane is the C═C bond) that can be modified to target various analytes, for in vitro and in vivo measurement of hydrazine. Prior to applying for sensing detection, the C═C bond connected to adamantane in ACL was first converted into dioxetane by irradiation to form the activated chemiluminescent AIEgen probe (ACLD). Then, the self-immolative reaction was triggered upon the deprotection of the acylated phenolic hydroxyl group in ACLD in the presence of hydrazine, resulting in the release of the high energy held in the 1,2-dioxetanes, and then, the chemiexcitation was triggered, thereby producing red chemiluminescence through the intramolecular chemiluminescence resonance energy transfer from Schaap's dioxetane to TPEDC. This chemiluminescent AIEgen probe was evaluated in a clean buffer environment as well as using living cells and mouse models.

Target MicroRNA-Responsive DNA Hydrogel-Based Surface-Enhanced Raman Scattering Sensor Arrays for MicroRNA-Marked Cancer Screening.

On the basis of a target microRNA (miRNA)-responsive DNA hydrogel, a novel surface-enhanced Raman scattering (SERS) sensor array with nine sensor units that can detect multiple cancer-related miRNAs in one sample was developed. The target miRNA-responsive DNA hydrogel was first formed in each sensor unit to realize the construction of the DNA hydrogel-based SERS sensor array. Initially, because of the blocking of the streptavidin (SA)-modified sensor units by the formed DNA hydrogel, the SERS tags (biotin/4-mercaptobenzonitrile-functionalized AuAg alloy nanoparticles (B/M-AuAgNPs)) could not pass through the hydrogel and bind to the SA-modified sensor surface; thus, obvious Raman signals could not be observed. After the introduction of the target miRNA, DNA hydrogels of the corresponding sensor unit were disintegrated accordingly, and SERS tags were able to pass through the hydrogel to be captured onto the SA-modified detection surface, thus resulting in strong Raman signals and the detection of target miRNA. The assay is validated under clean buffer conditions as well as in serum. This target miRNA-responsive DNA hydrogel-based SERS sensor array has attractive application prospects in cancer typing via blood miRNA measurements.

Two-Photon Excitation/Red Emission, Ratiometric Fluorescent Nanoprobe for Intracellular pH Imaging.

Herein, we describe a novel two-photon excitation/red emission-based ratiometric pH nanosensor consisting of a pH-sensitive two-photon dye and Tm3+-doped upconversion nanoparticles (UCNP). The fluorescence emission ratio between the dye (610 nm) and UCNPs (810 nm) (I610/I810) provides a linear indicator of pH values in the range from pH 4.0 to 6.5 with high sensitivity. These nanoprobes selectively accumulate in the lysosomes of cells, making them suitable for lysosomal pH tracking. This pH nanoprobe has been successfully applied in visualizing chemically stimulated changes of intracellular pH in living cells and tissues.

Alkyne/Ruthenium(II) Complex-Based Ratiometric Surface-Enhanced Raman Scattering Nanoprobe for In Vitro and Ex Vivo Tracking of Carbon Monoxide.

Here, we report a surface-enhanced Raman scattering (SERS) nanosensor for real-time ratiometric detection of carbon monoxide (CO) based on a ligand displacement mechanism. This nanoprobe consists of a gold-silver (Au-Ag) alloy nanoparticle core as the highly active SERS substrate, an alkyne/ruthenium(II) (alkyne/Ru(II)) complex immobilized on the surface as the CO-sensing element, and a porous silica shell to improve the stability and biocompatibility of the particle. Displacement of the alkyne ligand by CO results in a decrease of the alkyne vibrations and an increase of the metal carbonyl complex signals, thus allowing the effective ratiometric detection of CO in real-time. The great potential of this assay for CO detection is validated in clean buffer environments, live cells, and tissue slices.

An effector caspase Sp-caspase first identified in mud crab Scylla paramamosain exhibiting immune response and cell apoptosis.

Apoptosis plays a key role in the immune defense against pathogen infection, and caspase is one of the most important protease enzyme families, which could initiate and execute apoptosis. Among crustaceans, several caspase genes have been reported. However, caspase in mud crab Scylla paramamosain, have not been identified yet. Here, in the present study, we characterized a new caspase, named as Sp-caspase, from S. paramamosain. The full-length cDNA sequence of Sp-caspase contained 966 bp open reading frame, encoding 322 amino acids, and its molecular weight was 36 kDa. This gene has three conserved domains of the caspase family, a prodomain, a large subunit P20 and a small subunit P10. Phylogenetic analysis showed that Sp-caspase was clustered into an effector caspase group. Sp-caspase mainly distributed in midgut, hepatopancreas, hemocytes and female ovaries, and the transcript was significantly regulated in different tissues after being challenged with Vibrio parahaemolyticus, Vibrio alginolyticus or LPS. After infection with V. alginolyticus, the apoptosis rate of hemocytes notably increased, while the mRNA level of Sp-caspase and hydrolysis activity of caspase 3/7 significantly decreased. Furthermore, in vitro assays showed that the recombinant protein tSp-caspase (deletion of Sp-caspase prodomain) could efficiently recognize and cleave human caspase 3/7 substrate Ac-DEVD-pNA, functioning as an effector caspase. Meanwhile, heterologous expression of Sp-caspase in several cell lines (HEK293T cells, HeLa cells and HighFive cells) could specifically induce cell apoptosis. Taken together, these data demonstrated that Sp-caspase could perform apoptosis as an effector caspase. In addition, it might be a negative regulator of hemocytes apoptosis under pathogen infection, which would contribute to homeostasis and immune defense of hemocytes in S. paramamosain.

Nanoconjugates of Ag/Au/Carbon Nanotube for Alkyne-Meditated Ratiometric SERS Imaging of Hypoxia in Hepatic Ischemia.

We report a ratiometric surface-enhanced Raman scattering (SERS) nanoprobe for imaging hypoxic living cells or tissues, using azo-alkynes assembled on a single-walled carbon nanotube (SWCNT) surface-functionalized with Ag/Au alloy nanoparticles (SWCNT/Ag/AuNPs). Under a hypoxic condition, azobenzene derivatives preassembled on the surface of the nanostructures are reduced stepwise by various reductases and eventually removed from the surface of the SWCNT/Ag/AuNPs, resulting in the loss of characteristic alkyne Raman bands at 2207 cm-1. Using 2D-band of SWCNTs at 2578 cm-1 as the internal standard, we are able to determine the hypoxia level based on the ratio of two peak intensities ( I2578/ I2207) as demonstrated by the successful detection in different cell lines and rat liver tissue samples derived from hepatic ischemia surgery. By combining the outstanding anti-interference property of alkynes as SERS reporters and the distinct Raman responses of alkynes and SWCNTs in complex systems, this novel ratiometric SERS strategy holds promise in becoming a very useful tool for in vitro and in vivo monitoring of hypoxia in research and clinical settings.

Azoreductase-Responsive Nanoprobe for Hypoxia-Induced Mitophagy Imaging.

Mitophagy, as a crucial metabolic process, plays an essential role in maintaining cellular and tissue homeostasis. Various stresses especially hypoxia could improve intracellular reactive oxygen species (ROS) level to induce mitophagy. However, high-specific fluorescence imaging of mitophagy in living cells under hypoxia is still a challenge. Based on this, we report an azoreductase-responsive nanoprobe (termed Micelle@Mito-rHP@TATp, MCM@TATp) by encapsulating cationic spiropyrane derivative (Mito-rHP) to realize specific imaging of mitophagy in living cells under hypoxia. An azoreductase-responsive amphiphilic polymer, 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-azo- N-[maleimide(polyethylene glycol-2000) (Mal-PEG2000-Azo-DSPE), was first self-assembled into a micelle in aqueous solution. Meanwhile, the synthetic Mito-rHP encapsulated into this formed micelle to construct MCM. By modifying the surface of MCM with cell-penetrating peptide (TATp) to form MCM@TATp, the nanoprobe could avoid endolysosomal trapping. Under hypoxic conditions, the azobenzene moiety-contained MCM@TATp would be disrupted by the highly expressed azoreductase, then the encapsulated Mito-rHP would be released. Since Mito-rHP is a mitochondria-targeted and pH-sensitive probe, thus it could target into mitochondria and displayed a desirable "off-on" fluorescence response to mitophagy during which mitochondria were regarded to undergo acidification. The results indicated that the MCM@TATp in our design could image mitophagy under hypoxia in high-specificity. As further application, we have also demonstrated that this MCM@TATp can perform well to realize mitophagy imaging under the photodynamic therapy (PDT) which can induce hypoxia in treatment of cancer. We expect this new strategy would be a powerful tool for hypoxia-related fundamental and clinical research.

Azoreductase and Target Simultaneously Activated Fluorescent Monitoring for Cytochrome c Release under Hypoxia.

Hypoxia-induced cell apoptosis is closely related to degenerative diseases, autoimmune disorders, and tumor disease. In the process of apoptosis, the release of cytochrome c (Cyt c) is deemed to be a critical factor of the intrinsic pathway. Strategies for tracking Cyt c release in living cells based on the subcellular localization have been proposed recently. However, they are inherently lack of specificity for distinguishing the release of Cyt c in apoptotic process induced by hypoxia from other stimulus. In this paper, an azoreductase and target simultaneously activated fluorescent aptameric nanosensor integrating gold nanoparticles (AuNPs) and Cyt c-targeted aptamer-consisted double-stranded DNA hybridization complex (DSDHC) was proposed. It is worth noting that the employment of azobenzene moiety labeled on the DSDHC first ensured the aptameric nanosensor could be conjugated to the surface of AuNPs and then specifically reduced by hypoxia-related azoreductase. Upon Cyt c released from mitochondrion under hypoxia, the competitive displacement of Cyt c subsequently activated the fluorescence of the aptameric nanosensor and the fluorescence enhancement depended principally on the content of Cyt c release. Inspired by this, a new strategy for quantitative analysis and in situ imaging of Cyt c under hypoxic condition was proposed. The high spatial resolution monitoring of the dynamics of Cyt c release under hypoxia will offer a potentially rich opportunity to understand the apoptotic mechanism under hypoxic conditions, thus further facilitating risk assessment and risk reduction for hypoxic environments.

Molecular Engineering of α-Substituted Acrylate Ester Template for Efficient Fluorescence Probe of Hydrogen Polysulfides.

In this article, hydrogen polysulfide (H2Sn)-mediated Michael addition/cyclization cascade reactions toward acrylate ester analogues were exploited and utilized to construct novel and robust H2Sn-specific fluorescence probe for the first time. Through rational molecular engineering of the α-substituted acrylate ester template, the optimal candidate probe FP-CF3 containing trifluoromethyl-substituted acrylate ester group as recognition unit and 3-benzothiazol-7-hydroxycoumarin dye BHC as signal reporter can highly selectively detect H2Sn over other reactive sulfur species, especially biothiols including cysteine (Cys) and homocysteine (Hcy)/glutathione (GSH), with a rapid and significant turn-on fluorescence response (less than 60 s for response time and over 44-fold for signal-to-background ratio). The fast response and high selectivity of FP-CF3 for H2Sn could be attributed to a kinetically and spatially favored pentacyclic addition produced by the dual nucleophilic reaction of H2Sn with the CF3-substituted acrylate group. The big off-on fluorescence response is due to the pentacyclic intermediate results in the release of the highly fluorescent BHC. Moreover, it has been successfully applied in imaging of endogenous H2Sn fluctuation in living cells.

Alkyne-DNA-Functionalized Alloyed Au/Ag Nanospheres for Ratiometric Surface-Enhanced Raman Scattering Imaging Assay of Endonuclease Activity in Live Cells.

A novel ratiometric surface-enhanced Raman scattering (SERS) nanosensor has been developed to probe the activity of endonuclease under in vitro and in living cells conditions. The optimized alloyed Au/Ag nanoparticles (NPs) were synthesized as the SERS substrate, which combined the superior properties of both pure Au and pure Ag nanoparticles: they exhibit excellent plasmonic property with high chemical stability and low cytotoxicity. They were then employed for quantitative detection of endonuclease through functionalization with single-stranded DNA (ssDNA) carrying 3-[4-(phenylethynyl)benzylthio]propanoic acid (PEB) as endonuclease-responsive SERS signaling molecule and 4-thiophenylacetylene (TPA) as the internal standard SERS signaling molecule. In the presence of endonuclease, the ssDNA was cleaved, releasing PEB molecules from the particle surface and decreasing the SERS signal at 2215 cm-1 from PEB. Since the SERS signal at 1983 cm-1 from alkynyl TPA remained the same, quantitative detection of endonuclease was achieved, based on the ratiometric peak intensity of I1983/ I2215, with a detection limit as low as 0.056 unit/mL. A highly biocompatible and antijamming ratiometric SERS sensor was established by combining the alloyed Au/AgNPs with two unique alkynes molecules with Raman signals in the cellular silent region. The ratiometric sensor was successfully employed to detect intracellular endonuclease activity as well as endonuclease in living cells for the first time.

Upconversion Nanoprobes for the Ratiometric Luminescent Sensing of Nitric Oxide.

By taking advantage of the optical properties of upconversion nanoparticles (UCNPs), we have designed a luminescence ratiometric nanosensor for measuring nitric oxide (NO) in biological fluids, live cells, and tissues. This nanoconjugate consists of a UCNP core with two strong fluorescence emission peaks at 540 and 656 nm as the upconversion fluorophore, NO-reactive rhodamine B-derived molecules (RdMs) encapsulated within the mesopores of the mSiO2 shell, and a ß-cyclodextrin (ßCD) layer on the exterior of the particle. Reaction of the analyte with the O-phenylenediamine of the RdM induces opening of the spiro-ring and is accompanied by an appearance of a strong rhodamine B (RdB) absorption band between 500 and 600 nm, which has spectral overlap with the green emission (540 nm) of the UCNPs. This results in an increase in the I656/I540 ratio and quantitatively correlates with [NO]. The assay is validated under clean buffer conditions as well as inserum and liver tissue slices obtained from mouse models.

Noninvasive and Highly Selective Monitoring of Intracellular Glucose via a Two-Step Recognition-Based Nanokit.

Accurate determination of intracellular glucose is very important for exploring its chemical and biological functions in metabolism events of living cells. In this paper, we developed a new noninvasive and highly selective nanokit for intracellular glucose monitoring via two-step recognition. The liposome-based nanokit coencapsulated the aptamer-functionalized gold nanoparticles (AuNPs) and the Shinkai's receptor together. When the proposed nanokit was transfected into living cells, the Shinkai's receptor could recognize glucose first and then changed its conformation to endow aptamers with binding and sensing properties which were not readily accessible otherwise. Then, the binary complexes formed by the intracellular glucose and the Shinkai's receptor can in situ displace the complementary oligonucleotide of the aptamer on the surface of AuNPs. The fluorophore-labeled aptamer was away from the AuNPs, and the fluorescent state switched from "off" to "on". Through the secondary identification of aptamer, the selectivity of the Shinkai's receptor could be greatly improved while the intracellular glucose level was assessed by fluorescence signal recovery of aptamer. In the follow-up application, the approach exhibits excellent selectivity and is noninvasive for intracellular glucose monitoring under normoxia and hypoxia. To the best of our knowledge, this is the first time that the advantages of organic receptors and nucleic acids have been combined and highly selective monitoring of intracellular glucose has been realized via two-step recognition. We expect it to open up new possibilities to integrate devices for diagnosis of various metabolic diseases and insulin delivery.