Preparation of Protein-like Silver–Cysteine Hybrid Nanowires and Application in Ultrasensitive Immunoassay of Cancer Biomarker

Novel protein-like silver–cysteine hybrid nanowires (<i>p</i>-SCNWs) have been synthesized by a green, simple, nontemplate, seedless, and one-step aqueous-phase approach. AgNO₃ and l-cysteine were dissolved in distilled water, forming Ag–cysteine precipitates and HNO₃. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0 by addition of concentrated sodium hydroxide solution, leading to quick dissolution of the Ag-cysteine precipitates and sudden appearance of white precipitates of <i>p</i>-SCNWs. The <i>p</i>-SCNWs are monodispersed nanowires with diameter of 100 nm and length of tens of micrometers, and have abundant carboxyl (−COOH) and amine (−NH₂) groups at their surfaces, large amounts of peptide-linkages and S-bonding silver ions (Ag⁺) inside, making them look and act like Ag-hybrid protein nanostructures. The abundant −COOH and −NH₂ groups at the surfaces of <i>p</i>-SCNWs have been found to facilitate the reactions between the <i>p</i>-SCNWs and proteins including antibodies. Furthermore, the fact that the <i>p</i>-SCNWs contain large amounts of silver ions enables biofunctionalized <i>p</i>-SCNWs to be excellent signal amplifying chemiluminescence labels for ultrasensitive and highly selective detection of important antigens, such as cancer biomarkers. In this work, the immunoassay of carcinoembryonic antigen (CEA) in human serum was taken as an example to demonstrate the immunoassay applications of antibody-functionalized <i>p</i>-SCNWs. By the novel <i>p</i>-SCNW labels, CEA can be detected in the linear range from 5 to 400 fg/mL with a limit of detection (LOD) of 2.2 fg/mL (at signal-to-noise ratio of 3), which is much lower than that obtained by commercially available enzyme-linked immunosorbent assay (ELISA). Therefore, the synthesized <i>p</i>-SCNWs are envisioned to be an excellent carrier for proteins and related immunoassay strategy would have promising applications in ultrasensitive clinical screening of cancer biomarkers for early diagnostics of cancers

Encapsulation of Strongly Fluorescent Carbon Quantum Dots in Metal–Organic Frameworks for Enhancing Chemical Sensing

Novel highly fluorescent (FL) metal–organic frameworks (MOFs) have been synthesized by encapsulating branched poly-(ethylenimine)-capped carbon quantum dots (BPEI-CQDs) with a high FL quantum yield into the zeolitic imidazolate framework materials (ZIF-8). The as-synthesized FL-functionalized MOFs not only maintain an excellent FL activity and sensing selectivity derived from BPEI-CQDs but also can strongly and selectively accumulate target analytes due to the adsorption property of MOFs. The selective accumulation effect of MOFs can greatly amplify the sensing signal and specificity of the nanosized FL probe. The obtained BPEI-CQDs/ZIF-8 composites have been used to develop an ultrasensitive and highly selective sensor for Cu²⁺ ion, with a wide response range (2–1000 nM) and a very low detection limit (80 pM), and have been successfully applied in the detection of Cu²⁺ ions in environmental water samples. It is envisioned that various MOFs incorporated with FL nanostructures with high FL quantum yields and excellent selectivity would be designed and synthesized in similar ways and could be applied in sensing target analytes

Immobilization-Free Programmable Hairpin Probe for Ultrasensitive Electronic Monitoring of Nucleic Acid Based on a Biphasic Reaction Mode

This work designs a novel programmable hairpin probe (PHP) for the immobilization-free electrochemical detection of nucleic acid by coupling polymerase/nicking-induced isothermal signal amplification strategy with a biphasic reaction mode for the first time. The designed PHP (including a target-recognition region, a template sequence for enzymatic reaction and an inactivated <i>anti</i>-streptavidin aptamer) could program multiple isothermal reactions in the solution phase accompanying in situ amplified detectable signal at the electrode surface by the labeled ferrocene tag on the PHP. Upon addition of target analyte into the detection solution, target DNA initially hybridized with the recognition region on the PHP. Replication-induced strand-displacement generated an activated <i>anti</i>-streptavidin aptamer with the assistance of polymerase. Then, the polymerase/nicking enzymes could cleave and polymerize repeatedly the replication product, thus resulting in the formation of numerous template-complementary DNA initiator strands. The released initiator strands could retrigger the programmable hairpin probe to produce a large number of activated <i>anti</i>-streptavidin aptamers, which could be captured by the immobilized streptavidin on the electrode, thus activating the electrical contact between the labeled ferrocene and the electrode. Going after the aptamers, the carried ferrocene could produce the in situ amplified electronic signal within the applied potentials. Under optimal conditions, the electrochemical signal increased with the increasing target DNA concentration in the dynamic range from 5 fM to 10 pM with a detection limit (LOD) of 2.56 fM at the 3<i>s</i>_blank criterion. Importantly, the methodology with high specificity was also validated and evaluated by assaying 6 target DNA-spiked human serum and calf thymus DNA samples, and the recoveries were 95–110%. Further work for the programmable hairpin probe could be even utilized in a real world sample to detect miRNA-21 at femtomol level

Polyamine-Functionalized Carbon Quantum Dots as Fluorescent Probes for Selective and Sensitive Detection of Copper Ions

A novel sensing system has been designed for Cu²⁺ ion detection based on the quenched fluorescence (FL) signal of branched poly­(ethylenimine) (BPEI)-functionalized carbon quantum dots (CQDs). Cu²⁺ ions can be captured by the amino groups of the BPEI-CQDs to form an absorbent complex at the surface of CQDs, resulting in a strong quenching of the CQDs’ FL via an inner filter effect. Herein, we have demonstrated that this facile methodology can offer a rapid, reliable, and selective detection of Cu²⁺ with a detection limit as low as 6 nM and a dynamic range from 10 to 1100 nM. Furthermore, the detection results for Cu²⁺ ions in a river water sample obtained by this sensing system agreed well with that by inductively couple plasma mass spectrometry, suggesting the potential application of this sensing system

Carbon Quantum Dot-Functionalized Aerogels for NO₂ Gas Sensing

Silica aerogels functionalized with strongly fluorescent carbon quantum dots were first prepared and used for simple, sensitive, and selective sensing of NO₂ gas. In the presence of ethanol, homemade silica aerogels with a large specific surface area of 801.17 m²/g were functionalized with branched polyethylenimine-capped quantum dots (BPEI-CQDs) with fluorescence quantum yield higher than 40%. The prepared porous CQD-aerogel hybrid material could maintain its excellent fluorescence (FL) activity in its solid state. The FL of CQD-aerogel hybrid material could be selectively and sensitively quenched by NO₂ gas, suggesting a promising application of the new FL-functionalized aerogels in gas sensing

Simultaneous Multiplexed Stripping Voltammetric Monitoring of Marine Toxins in Seafood Based on Distinguishable Metal Nanocluster-Labeled Molecular Tags

Marine toxins from microscopic algae can accumulate through the food chain and cause various neurological and gastrointestinal illnesses for human health. Herein, we designed a new ultrasensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of brevetoxin B (BTX-2) and dinophysistoxin-1 (DTX-1) in seafood using distinguishable metal nanocluster-labeled molecular tags as traces on bifunctionalized magnetic capture probes. To construct such a bifunctionalized probe, monoclonal mouse anti-BTX-2 (mAb₁) and anti-DTX-1 (mAb₂) antibodies were co-immobilized on a magnetic bead (MB–mAb_1,2). The distinguishable metal nanoclusters including cadmium nanoclusters (CdNC) and copper nanoclusters (CuNC) were synthesized using the artificial peptides with amino acid sequence CCCYYY, which were used as distinguishable signal tags for the label of the corresponding bovine serum albumin–BTX-2 and bovine serum albumin–DTX-1 conjugates. A competitive-type immunoassay format was adopted for the online simultaneous monitoring of BTX-2 and DTX-1 on a homemade flow-through magnetic detection cell. The assay was based on the stripping voltammetric behaviors of the labeled CdNC and CuNC at the various peak potentials in pH 2.5 HCl containing 0.01 M KCl using square wave anodic stripping voltammetry (SWASV). Under optimal conditions, the multiplexed immunoassays enabled simultaneous detection of BTX-2 and DTX-1 in a single run with wide working ranges of 0.005–5 ng mL^–1 for two marine toxins. The limit of detection (LOD) and limit of quantification (LOQ) were 1.8 and 6.0 pg mL^–1 for BTX-2, while those for DTX-1 were 2.2 and 7.3 pg mL^–1, respectively. No non-specific adsorption and electrochemical cross-talk between neighboring sites were observed during a series of procedures to detect target analytes. The covalent conjugation of biomolecules onto the nanoclusters and magnetic beads resulted in good repeatability and intermediate precision down to 9.5%. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of 12 spiked samples, including Sinonovacula constricta, Musculista senhousia, and Tegillarca granosa, between the multiplexed immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for analysis of BTX-2 and DTX-1

Graphene Quantum Dots/l‑Cysteine Coreactant Electrochemiluminescence System and Its Application in Sensing Lead(II) Ions

A new coreactant electrochemiluminescence (ECL) system including single-layer graphene quantum dots (GQDs) and l-cysteine (l-Cys) was found to be able to produce strong cathodic ECL signal. The ECL signal of GQD/l-Cys coreactant system was revealed to be mainly dependent on some key factors, including the oxidation of l-Cys, the presence of dissolved oxygen and the reduction of GQDs. Then, a possible ECL mechanism was proposed for the coreactant ECL system. Furthermore, the ECL signal of the GQD/l-Cys system was observed to be quenched by lead­(II) ions (Pb²⁺). After optimization of some important experimental conditions, including concentrations of GQDs and l-Cys, potential scan rate, response time, and pH value, an ECL sensor was developed for the detection of Pb²⁺. The new methodology can offer a rapid, reliable, and selective detection of Pb²⁺ with a detection limit of 70 nM and a dynamic range from 100 nM to 10 μM

Gold Nanoparticle-Graphite-Like C₃N₄ Nanosheet Nanohybrids Used for Electrochemiluminescent Immunosensor

Two-dimensional graphite-like carbon nitride nanosheets (g-C₃N₄ NSs) were hybridized with gold nanoparticles (Au NPs) to construct an electrochemiluminescence (ECL) immunosensor. The prepared Au NP-functionalized g-C₃N₄ NS nanohybrids (Au-g-C₃N₄ NHs) exhibit strong and stable cathodic ECL activity compared to g-C₃N₄ NSs due to the important roles of Au NPs in trapping and storing the electrons from the conduction band of g-C₃N₄ NSs, as well as preventing high energy electron-induced passivation of g-C₃N₄ NSs. On the basis of the improved ECL stability and ECL peak intensity of the Au-g-C₃N₄ NHs, a novel ECL immunosensor was developed to detect carcinoembryonic antigen (CEA) as a model target analyte. The ECL immunosensor has a sensitive response to CEA in a linear range of 0.02–80 ng mL^–1 with a detection limit of 6.8 pg mL^–1. Additionally, the proposed immunosensor shows high specificity, good reproducibility, and long-term stability

DNA-Based Hybridization Chain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection of Proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL^–1. Importantly, the methodology can be further extended to the detection of other proteins or biomarkers

Turn-On Fluorescence Sensor for Intracellular Imaging of Glutathione Using g‑C₃N₄ Nanosheet–MnO₂ Sandwich Nanocomposite

Herein, a novel fluorescence sensor based on g-C₃N₄ nanosheet–MnO₂ sandwich nanocomposite has been developed for rapid and selective sensing of glutathione (GSH) in aqueous solutions, as well as living cells. The graphitic-phase C₃N₄ (g-C₃N₄) nanosheet used here is a new type of carbon-based nanomaterial with high fluorescence quantum yield and high specific surface area. We demonstrate a facile one-step approach for the synthesis of a g-C₃N₄ nanosheet–MnO₂ sandwich nanocomposite for the first time. The fluorescence of g-C₃N₄ nanosheet in this nanocomposite is quenched, which attributing to fluorescence resonance energy transfer (FRET) from a g-C₃N₄ nanosheet to the deposited MnO₂. Upon the addition of GSH, MnO₂ is reduced to Mn²⁺, which leads to the elimination of FRET. As a result, the fluorescence of g-C₃N₄ nanosheet is restored. Importantly, the chemical response of the g-C₃N₄–MnO₂ nanocomposite exhibits great selectivity toward GSH relative to other electrolytes and biomolecules. Under the optimal conditions, the detection limit of 0.2 μM for GSH in aqueous solutions can be reached. Furthermore, the g-C₃N₄–MnO₂ nanocomposite is confirmed to be membrane-permeable and have low cytotoxicity. Moreover, we successfully apply this sensor for visualizing and monitoring change of the intracellular GSH in living cells. Moreover, the proposed sensor shows satisfying performance, such as low cost, easy preparation, rapid detection, good biocompatibility, and turn-on fluorescence response