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

Copper-Based Metal–Organic Framework Nanoparticles with Peroxidase-Like Activity for Sensitive Colorimetric Detection of <i>Staphylococcus aureus</i>

Cu-MOF nanoparticles with an average diameter of 550 nm were synthesized from 2-aminoterephthalic acid and Cu­(NO₃)₂ by a mixed solvothermal method. The Cu-MOF nanoparticles can show peroxidase-like activity that can catalyze 3,3′,5,5′-tetramethylbenzidine to produce a yellow chromogenic reaction in the presence of H₂O₂. The presence of abundant amine groups on the surfaces of Cu-MOF nanoparticles enables facile modification of <i>Staphylococcus aureus</i> (<i>S. aureus</i>) aptamer on Cu-MOF nanoparticles. By combining Cu-MOF-catalyzed chromogenic reaction with aptamer recognition and magnetic separation, a simple, sensitive, and selective colorimetric method for the detection of <i>S. aureus</i> was developed

Fluorescent Immunoassay for the Detection of Pathogenic Bacteria at the Single-Cell Level Using Carbon Dots-Encapsulated Breakable Organosilica Nanocapsule as Labels

Herein, carbon dots (CDs)-encapsulated breakable organosilica nanocapsules (BONs) were facilely prepared and used as advanced fluorescent labels for ultrasensitive detection of Staphylococcus aureus. The CDs were entrapped in organosilica shells by cohydrolyzation of tetraethyl orthosilicate and bis­[3-(triethoxysilyl)­propyl]­disulfide to form core–shell CDs@BONs, where hundreds of CDs were encapsulated in each nanocapsule. Immunofluorescent nanocapsules, i.e., anti-S. aureus antibody-conjugated CDs@BONs, were prepared to specifically recognize S. aureus. Before fluorescent detection, CDs were released from the BONs by simple NaBH₄ reduction. The fluorescent signals were amplified by 2 orders of magnitude because of hundreds of CDs encapsulated in each nanocapsule, compared with a conventional immunoassay using CDs as fluorescent labels. A linear range was obtained at the S. aureus concentration from 1 to 200 CFU mL^–1. CDs@BONs are also expected to expand to other systems and allow the detection of ultralow concentrations of targets

Carbon Nanotube-Polyamidoamine Dendrimer Hybrid-Modified Electrodes for Highly Sensitive Electrochemical Detection of MicroRNA24

A simple and ultrasensitive microRNA (miRNA) electrochemical biosensor employing multiwalled carbon nanotube (MWCNT)-polyamidoamine (PAMAM) dendrimer and methylene blue (MB) redox indicator is reported in this work. The assay utilizes a glass carbon (GC) electrode modified with MWCNT-PAMAM, on which the oligonucleotide capture probes are immobilized. The electrochemical detection of miRNAs is completed by measuring the reduction signal change of MB before and after the probe hybridization with target miRNA (miRNA24 is used as a model case). The MWCNT-PAMAM/GC electrode shows greatly enhanced signal to MB reduction in contrast to bare GC electrode. The functionalization of MWCNT with PAMAM maintains the electrochemical property of MWCNT to MB reduction but minimizes the undesired adsorption of MB on the MWCNT surface. The effect of experimental variables on the miRNA detection is investigated and optimized. A detection limit of 0.5 fM and a linear peak current density-concentration relationship up to 100 nM are obtained following 60 min hybridization. The proposed assay is successfully used to detect miRNA24 in total RNA sample extracted from HeLa cells

Boronic Acid-Decorated Carbon Dot-Based Semiselective Multichannel Sensor Array for Cytokine Discrimination and Oral Cancer Diagnosis

Cytokines are essential components of the immune system and are recognized as significant biomarkers. However, detection of a single cytokine is not precise and reliable enough to satisfy the requirements for diagnosis. Herein, we developed a pattern recognition-based method for the multiplexed sensing of cytokines, which involves three-color-emitting boronic acid-decorated carbon dots (BCDs) and arginine-modified titanium carbide (Ti3C2 MXenes) as the sensor array. Initially, the fluorescence signals of the three BCDs were quenched by Ti3C2 MXenes. In the presence of cytokines, the fluorescence intensity of the BCDs was restored or further quenched by different cytokines. The fluorescence response occurs in two steps: first, boronic acid interacts with cis-diol functional groups of cytokines, and second, arginine headgroup selectively interacts with glycans. By exploiting the different competing binding of the BCDs and the cytokines toward Ti3C2 MXenes, seven cytokines and their mixtures can be effectively discriminated at a concentration of 20 ng mL–1. Furthermore, our sensor array demonstrated an excellent performance in classifying human oral cancer saliva samples from healthy individuals with clinically relevant specificity. The noninvasive method offers a rapid approach to cytokine analysis, benefiting early and timely clinical diagnosis and treatment

Multifunctional Electrochemical Platforms Based on the Michael Addition/Schiff Base Reaction of Polydopamine Modified Reduced Graphene Oxide: Construction and Application

In this paper, a new strategy for the construction of multifunctional electrochemical detection platforms based on the Michael addition/Schiff base reaction of polydopamine modified reduced graphene oxide was first proposed. Inspired by the mussel adhesion proteins, 3,4-dihydroxyphenylalanine (DA) was selected as a reducing agent to simultaneously reduce graphene oxide and self-polymerize to obtain the polydopamine-reduced graphene oxide (PDA-rGO). The PDA-rGO was then functionalized with thiols and amines by the reaction of thiol/amino groups with quinine groups of PDA-rGO via the Michael addition/Schiff base reaction. Several typical compounds containing thiol and/or amino groups such as 1-[(4-amino)­phenylethynyl] ferrocene (Fc-NH₂), cysteine (cys), and glucose oxidase (GOx) were selected as the model molecules to anchor on the surface of PDA-rGO using the strategy for construction of multifunctional electrochemical platforms. The experiments revealed that the composite grafted with ferrocene derivative shows excellent catalysis activity toward many electroactive molecules and could be used for individual or simultaneous detection of dopamine hydrochloride (DA) and uric acid (UA), or hydroquinone (HQ) and catechol (CC), while, after grafting of cysteine on PDA-rGO, simultaneous discrimination detection of Pb²⁺ and Cd²⁺ was realized on the composite modified electrode. In addition, direct electron transfer of GOx can be observed when GOx-PDA-rGO was immobilized on glassy carbon electrode (GCE). When glucose was added into the system, the modified electrode showed excellent electric current response toward glucose. These results inferred that the proposed multifunctional electrochemical platforms could be simply, conveniently, and effectively regulated through changing the anchored recognition or reaction groups. This study would provide a versatile method to design more detection or biosensing platforms through a chemical reaction strategy in the future

Nanosensor Composed of Nitrogen-Doped Carbon Dots and Gold Nanoparticles for Highly Selective Detection of Cysteine with Multiple Signals

Biological thiols play a critical role in biological processes and are involved in a variety of diseases. The discrimination detection of biological thiols is of increasing importance in clinical diagnosis. In this paper, a novel nanosensor was developed to discriminate cysteine (Cys) from homocysteine (Hcy) and glutathione (GSH) with multiple signals: colorimetric, photoluminescence (PL), and up-conversional photoluminescence (UCP). The nanosensor (NC-dots/AuNPs) was constructed by nitrogen-doped carbon dots (NC-dots) and gold nanoparticles (AuNPs) through assembling NC-dots “shell” on AuNPs and showed the obvious different response to Cys, Hcy, and GSH with colorimetric, PL, and UCP signals. The discrimination effect for Cys is originated from conformations and interaction difference of the thiols groups in Cys and Hcy and/or GSH with AuNPs. Among them, only Cys can quickly penetrate into the NC-dots “shell” of the composite and induce the dispersing of the aggregated NC-dots/AuNPs, which lead to the color change from purple to red and the recovery of PL and UCP of NC-dots. This assay was successfully applied for the detection of Cys in human serum with the detection limit of 4 nM

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

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