Non-Redox Modulated Fluorescence Strategy for Sensitive and Selective Ascorbic Acid Detection with Highly Photoluminescent Nitrogen-Doped Carbon Nanoparticles via Solid-State Synthesis

Highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) were prepared by a simple and green route employing sodium alginate as a carbon source and tryptophan as both a nitrogen source and a functional monomer. The as-synthesized N-CNPs exhibited excellent water solubility and biocompatibility with a fluorescence quantum yield of 47.9%. The fluorescence of the N-CNPs was intensively suppressed by the addition of ascorbic acid (AA). The mechanism of the fluorescence suppression of the N-CNPs was investigated, and the synergistic action of the inner filter effect (IFE) and the static quenching effect (SQE) contributed to the intensive fluorescence suppression, which was different from those reported for the traditional redox-based fluorescent probes. Owing to the spatial effect and hydrogen bond between the AA and the groups on the N-CNP surface, excellent sensitivity and selectivity for AA detecting was obtained in a wide linear relationship from 0.2 μM to 150 μM. The detection limit was as low as 50 nM (signal-to-noise ratio of 3). The proposed sensing systems also represented excellent sensitivity and selectivity for AA analysis in human biological fluids, providing a valuable platform for AA sensing in clinic diagnostic and drug screening

Eu,Sm,Mn-Doped CaS Nanoparticles with 59.3% Upconversion-Luminescence Quantum Yield: Enabling Ultrasensitive and Facile Smartphone-Based Sulfite Detection

Eu,Sm,Mn-doped CaS (ESM-CaS) nanoparticles demonstrate a remarkable upconversion luminescence (UCL) efficiency with a quantum yield of nearly 60%, enabling many new applications and devices. We describe an ESM-CaS nanoparticle-based paper test strip for one-shot quantitative measurement of sulfite concentration using a smartphone-based reader. The integrated UCL-based sulfite detection system features high sensitivity and facile operation without the need for separation and pretreatment. Moreover, the design principles are general in nature and so can be tailored for the detection and quantification of a variety of other analytes

Graphene Oxide–Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)–peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP–dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening

Aptameric Peptide for One-Step Detection of Protein Kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP₂₀) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni­(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL^–1 and a linear range from 0.64 to 22.33 mU μL^–1. This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate

Versatile Electrochemiluminescent Biosensor for Protein–Nucleic Acid Interaction Based on the Unique Quenching Effect of Deoxyguanosine-5′-phosphate on Electrochemiluminescence of CdTe/ZnS Quantum Dots

In this paper, the efficient quenching effect of deoxyguanosine-5′-phosphate (dGMP) on anodic electrochemiluminescence (ECL) of the CdTe/ZnS quantum dots (QDs) is reported for the first time. This ECL quenching was found to be specific for free dGMP and not observed for dGMP residues in different DNA structures. The unique dGMP-based QDs ECL quenching was then utilized to develop a versatile biosensing strategy to determine various protein–DNA interactions with the assistance of exonuclease, Exo I, to hydrolyze DNA and liberate dGMP. Taking single-stranded DNA binding protein (SSB) and thrombin as examples, two novel detection modes have been developed based on dGMP–QDs ECL strategy. The first method used hairpin probes and SSB-promoted probe cleavage by Exo I for facile signal-off detection of SSB, with a wide linear range of 1–200 nM and a low detection limit of 0.1 nM. The second method exploited aptamer–thrombin binding to protect probes against Exo I degradation for sensitive signal-on detection of thrombin, giving a linear response over a range of 1–150 nM and a detection limit as low as 0.1 nM. Both methods were homogeneous and label-free without QDs or DNA modification. Therefore, this dGMP-specific QDs ECL quenching presents a promising detection mechanism suitable for probing various protein–nucleic acid interactions

Enzyme-Activated G‑Quadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis

Fluorogenic probes targeting G-quadruplex structures have emerged as the promising toolkit for functional research of G-quadruplex and biosensor development. However, their biosensing applications are still largely limited in in-tube detection. Herein, we proposed a fluorescent bioimaging method based on enzyme-generated G-quadruplexes for detecting apoptotic cells at the cell and tissue level, namely, terminal deoxynucleotidyl transferase (TdT)-activated de novo G-quadruplex synthesis (TAGS) assay. The detection target is genomic DNA fragmentation, a biochemical hallmark of apoptosis. The TAGS assay can efficiently “tag” DNA fragments via using their DNA double-strand breaks (DSBs) to initiate the de novo synthesis of G-quadruplexes by TdT with an unmodified G-rich dNTP pool, followed by a rapid fluorescent readout upon the binding of thioflavin T (ThT), a fluorogenic dye highly specific for G-quadruplex. The feasibility of the TAGS assay was proved by in situ sensitive detection of individual apoptotic cells in both cultured cells and tissue sections. The TAGS assay has notable advantages, including being label-free and having quick detection, high sensitivity and contrast, mix-and-read operation without tedious washing, and low cost. This method not only shows the feasibility of G-quadruplex in tissue bioanalysis but also provides a promising tool for basic research of apoptosis and drug evaluation for antitumor therapy

Time-Resolved Luminescence Biosensor for Continuous Activity Detection of Protein Acetylation-Related Enzymes Based on DNA-Sensitized Terbium(III) Probes

Protein acetylation of histone is an essential post-translational modification (PTM) mechanism in epigenetic gene regulation, and its status is reversibly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Herein, we have developed a sensitive and label-free time-resolved luminescence (TRL) biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb³⁺/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb³⁺ luminescence as the output signal, the polycationic substrate peptides interact with DNA with high affinity and subsequently replace Tb³⁺, eliminating the luminescent signal. HAT-catalyzed acetylation remarkably reduces the positive charge of the peptides and diminishes the peptide/DNA interaction, resulting in the signal on detection via recovery of DNA-sensitized Tb³⁺ luminescence. With this TRL sensor, HAT (p300) can be sensitively detected with a wide linear range from 0.2 to 100 nM and a low detection limit of 0.05 nM. The proposed sensor was further used to continuously monitor the HAT activity in real time. Additionally, the TRL biosensor was successfully applied to evaluating HAT inhibition by two specific inhibitors, anacardic acid and C464, and satisfactory <i>Z</i>′-factors above 0.73 were obtained. Moreover, this sensor is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation with a linear range from 0.5 to 500 nM and a detection limit of 0.5 nM. The proposed sensor is a convenient, sensitive, and mix-and-read assay, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery

Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free Detection of Copper(II) Ion and Acetylcholinesterase Activity

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H₃₉GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H₃₉GFP as the signal reporter, a label-free fluorometric sensor for Cu²⁺ sensing was developed based on the unique multivalent metal ion-binding property of H₃₉GFP and fluorescence quenching effect of Cu²⁺ by electron transfer. The high affinity of H₃₉GFP with Cu²⁺ (<i>K</i>_d, 16.2 nM) leads to rapid detection of Cu²⁺ in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H₃₉GFP/Cu²⁺ complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu²⁺ and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL^–1) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H₃₉GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms

Phospholipid-Tailored Titanium Carbide Nanosheets as a Novel Fluorescent Nanoprobe for Activity Assay and Imaging of Phospholipase D

As one of the emerging inorganic graphene analogues, two-dimensional titanium carbide (Ti₃C₂) nanosheets have attracted extensive attention in recent years because of their remarkable structural and electronic properties. Herein, a sensitive and selective nanoprobe to fluorescently probe phospholipase D activity was developed on the basis of an ultrathin Ti₃C₂ nanosheets-mediated fluorescence quenching effect. Ultrathin Ti₃C₂ nanosheets with ∼1.3 nm in thickness were synthesized from bulk Ti₃AlC₂ powder by a two-step exfoliation procedure and further modified by a natural phospholipid that is doped with rhodamine B-labeled phospholipid (RhB-PL-Ti₃C₂). The close proximity between RhB and Ti₃C₂ leads to efficient fluorescence quenching (>95%) of RhB by energy transfer. Phospholipase D-catalyzed lipolysis of the phosphodiester bond in RhB-PL results in RhB moving away from the surface of Ti₃C₂ nanosheets and subsequent fluorescence recovery of RhB, providing a fluorescent “switch-on” assay for the phospholipase D activity. The proposed nanoprobe was successfully applied to quantitatively determine phospholipase D activity with a low limit of detection (0.10 U L^–1) and to measure its inhibition. Moreover, in situ monitoring and imaging the activity of phospholipase D in living cells were achieved using this biocompatible nanoprobe. These results reveal that Ti₃C₂ nanosheets-based probes exhibit great potential in fluorometric assay and clinical diagnostic applications

Self-Assembled DNA Hydrogel Based on Enzymatically Polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction

DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system