Visual Biopsy by Hydrogen Peroxide-Induced Signal Amplification

Visual biopsy has attracted special interest by surgeons due to its simplicity and practicality; however, the limited sensitivity of the technology makes it difficult to achieve an early diagnosis. To circumvent this problem, herein, we report a visual signal amplification strategy for establishing a marker-recognizable biopsy that enables early cancer diagnosis. In our proposed approach, hydrogen peroxide (H₂O₂) was selected as a potential underlying marker for its compact relationship in cancer progression. For selective recognition of H₂O₂ in the process of visual biopsy, a benzylbenzeneboronic acid pinacol ester-decorated copolymer, namely, PMPC–Bpe, was synthesized, affording the final formation of the H₂O₂-responsive micelles in which amylose was trapped. The presence of H₂O₂ activates the boronate ester recognition site and induces it releasing abundant indicator amylose, leading to signal amplification. The indicator came across the solution of KI/I₂ added to the sample, and the formative amylose–KI/I₂ complex has a distinct blue color at 574 nm for visual amplification detection. The feasibility of the proposed method is demonstrated by visualizing the H₂O₂ content of cancer at different stages and three kinds of actual cancerous samples. As far as we know, this is the first paradigm to rationally design a signaling amplification-based molecular recognizable biopsy for visual and sensitive disease identification, which will extend new possibilities for marker-recognition and signal amplification-based biopsy in disease progressing

A Reversible Nanolamp for Instantaneous Monitoring of Cyanide Based on an Elsner-Like Reaction

It is well-known that cyanide ion (CN^–) is a hypertoxic anion, which can cause adverse effects in both the environment and living beings; thus, it is highly desirable to develop strategies for detecting CN^–, especially in water and food. However, due to the short half-life of free cyanide, long analysis time and/or interference from other competitive ions are general challenges for accurate monitoring of CN^–. In this work, through the investigation on the sequence-dependent optical interaction of DNA-CuNPs with the fluorophore (e.g., EBMVC-B), we found, for the first time, that DNA-CuNPs were an ideal alternative as fluorescence quencher in constructing a sensor which could be illuminated by CN^– based on an Elsner-like reaction and that the signal switching was dependent on poly­(AT/TA) dsDNA sequence. By virtue of CuNPs’ small size and its high chemical reactivity with cyanide, the lighting of fluorescence was ultrarapid and similar to the hairtrigger “turn-on” of a lamp, which is significant for accurately monitoring a target of short half-life (e.g., cyanide). Attributed to the unique Elsner-like reaction between CN^– and the Cu atoms, high selectivity was achieved for CN^– monitoring by the nanolamp, with practical applications in real water and food samples. In addition, because of the highly efficient <i>in situ</i> formation of DNA-CuNPs and the approximative stoichiometry between CN^– and Cu²⁺ in the fluorescence switching, the nanolamp could be reversibly turned on and off through the alternate regulation of CN^– and Cu²⁺, displaying potential for developing reusable nanosensors and constructing optical molecular logic circuits

Direct Detection of Nucleic Acid with Minimizing Background and Improving Sensitivity Based on a Conformation-Discriminating Indicator

As is well-known, the nucleic acid indicator-based strategy is one of the major approaches to monitor the nucleic acid hybridization-mediated recognition events in biochemical analysis, displaying obvious advantages including simplicity, low cost, convenience, and generality. However, conventional indicators either hold strong self-fluorescence or can be lighted by both ssDNA and dsDNA, lacking absolute selectivity for a certain conformation, always with high background interference and low sensitivity in sensing; and additional processing (e.g., nanomaterial-mediated background suppression, and enzyme-catalyzed signal amplification) is generally required to improve the detection performance. In this work, a carbazole derivative, EBCB, has been synthesized and screened as a dsDNA-specific fluorescent indicator. Compared with conventional indicators under the same conditions, EBCB displayed a much higher selective coefficient for dsDNA, with little self-fluorescence and negligible effect from ssDNA. Based on its superior capability in DNA conformation-discrimination, high sensitivity with minimizing background interference was demonstrated for direct detection of nucleic acid, and monitoring nucleic acid-based circuitry with good reversibity, resulting in low detection limit and high capability for discriminating base-mismatching. Thus, we expect that this highly specific DNA conformation-discriminating indicator will hold good potential for application in biochemical sensing and molecular logic switching

Poly(thymine)-Templated Copper Nanoparticles as a Fluorescent Indicator for Hydrogen Peroxide and Oxidase-Based Biosensing

Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly­(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H₂O₂ and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly­(thymine) length-dependent formation of T-CuNPs and the probe’s oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast <i>in situ</i> manner in the absence of H₂O₂, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H₂O₂, leading to the decline of fluorescence intensity. By taking advantage of H₂O₂ as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications

A Target-Lighted dsDNA-Indicator for High-Performance Monitoring of Mercury Pollution and Its Antagonists Screening

As well-known, the excessive discharge of heavy-metal mercury not only destroys the ecological environment, bust also leads to severe damage of human health after ingestion via drinking and bioaccumulation of food chains, and mercury ion (Hg²⁺) is designated as one of most prevalent toxic metal ions in drinking water. Thus, the high-performance monitoring of mercury pollution is necessary. Functional nucleic acids have been widely used as recognition probes in biochemical sensing. In this work, a carbazole derivative, ethyl-4-[3,6-bis­(1-methyl-4-vinylpyridium iodine)-9H-carbazol −9-yl)] butanoate (EBCB), has been synthesized and found as a target-lighted DNA fluorescent indicator. As a proof-of-concept, Hg²⁺ detection was carried out based on EBCB and Hg²⁺-mediated conformation transformation of a designed DNA probe. By comparison with conventional nucleic acid indicators, EBCB held excellent advantages, such as minimal background interference and maximal sensitivity. Outstanding detection capabilities were displayed, especially including simple operation (add-and-read manner), ultrarapidity (30 s), and low detection limit (0.82 nM). Furthermore, based on these advantages, the potential for high-performance screening of mercury antagonists was also demonstrated by the fluorescence change of EBCB. Therefore, we believe that this work is meaningful in pollution monitoring, environment restoration and emergency treatment, and may pave a way to apply EBCB as an ideal signal transducer for development of high-performance sensing strategies

Quantitative Monitoring of Hypoxia-Induced Intracellular Acidification in Lung Tumor Cells and Tissues Using Activatable Surface-Enhanced Raman Scattering Nanoprobes

Hypoxia is considered to contribute to pathophysiology in various cells and tissues, and a clear understanding about the relationship between hypoxia and intracellular acidification will help to elucidate the complex mechanism of glycolysis under hypoxia. However, current studies are mainly focused on overexpression of intracellular reductases accelerated by hypoxia, and the investigations focusing on the relationship between hypoxic degree and intracellular acidification remain to be explored. For this vacuity, we report herein a new activatable nanoprobe for sensing pH change under different degrees of hypoxia by surface-enhanced Raman spectroscopy (SERS). The monitoring was based on the SERS spectra changes of 4-nitrothiophenol (4-NTP)-functionalized gold nanorods (AuNR@4-NTP) resulting from the nitroreductase (NTR)-triggered reduction under hypoxic conditions while the as-generated 4-aminothiophenol (4-ATP) is a pH-sensitive molecule. This unique property can ensure the SERS monitoring of intracellular acidification in living cells and tissues under hypoxic conditions. Dynamic pH analysis indicated that the pH decreased from 7.1 to 6.5 as a function of different degrees of hypoxia (from 15 to 1%) due to excessive glycolytic activity triggered by hypoxia. Given the known advantages of SERS sensing, these findings hold promise in studies of pathophysiological pathways involving hypoxia

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

In this article, hydrogen polysulfide (H₂S_<i>n</i>)-mediated Michael addition/cyclization cascade reactions toward acrylate ester analogues were exploited and utilized to construct novel and robust H₂S_<i>n</i>-specific fluorescence probe for the first time. Through rational molecular engineering of the α-substituted acrylate ester template, the optimal candidate probe <b>FP–CF</b>_<b>3</b> containing trifluoromethyl-substituted acrylate ester group as recognition unit and 3-benzothiazol-7-hydroxycoumarin dye <b>BHC</b> as signal reporter can highly selectively detect H₂S_<i>n</i> 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 <b>FP–CF</b>_<b>3</b> for H₂S_<i>n</i> could be attributed to a kinetically and spatially favored pentacyclic addition produced by the dual nucleophilic reaction of H₂S_<i>n</i> 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 <b>BHC</b>. Moreover, it has been successfully applied in imaging of endogenous H₂S_<i>n</i> fluctuation in living cells

A Reaction-Based Ratiometric Bioluminescent Platform for Point-of-Care and Quantitative Detection Using a Smartphone

Fluorescent probes have emerged as powerful tools for the detection of different analytes by virtue of structural tenability. However, the requirement of an excitation source largely hinders their applicability in point-of-care detection, as well as causing autofluorescence interference in complex samples. Herein, based on bioluminescence resonance energy transfer (BRET), we developed a reaction-based ratiometric bioluminescent platform, which allows the excitation-free detection of analytes. The platform has a modular design consisting of a NanoLuc-HaloTag fusion as an energy donor, to which a synthetic fluorescent probe is bioorthogonally labeled as recognition moiety and energy acceptor. Once activated by the target, the fluorescent probe can be excited by NanoLuc to generate a remarkable BRET signal, resulting in obvious color changes of luminescence, which can be easily recorded and quantitatively analyzed by a smartphone. As a proof of concept, a fluorescent probe for HOCl was synthesized to construct the bioluminescent system. Results demonstrated the system showed a constant blue/red emission ratio which is independent to the signal intensity, allowing the quantification of HOCl concentration with high sensitivity (limit of detection (LOD) = 13 nM) and accuracy. Given the universality, this reaction-based bioluminescent platform holds great potential for point-of-care and quantitative detection of reactive species

Two-Photon Sensing and Imaging of Endogenous Biological Cyanide in Plant Tissues Using Graphene Quantum Dot/Gold Nanoparticle Conjugate

One main source of cyanide (CN^–) exposure for mammals is through the plant consumption, and thus, sensitive and selective CN^– detection in plants tissue is a significant and urgent work. Although various fluorescence probes have been reported for CN^– in water and mammalian cells, the detection of endogenous biological CN^– in plant tissue remains to be explored due to the high background signal and large thickness of plant tissue that hamper the effective application of traditional one-photo excitation. To address these issues, we developed a new two-photo excitation (TPE) nanosensor using graphene quantum dots (GQDs)/gold nanoparticle (AuNPs) conjugate for sensing and imaging endogenous biological CN^–. With the benefit of the high quenching efficiency of AuNPs and excellent two-photon properties of GQDs, our sensing system can achieve a low detection limit of 0.52 μM and deeper penetration depth (about 400 μm) without interference from background signals of a complex biological environment, thus realizing sensing and imaging of CN^– in different types of plant tissues and even monitoring CN^– removal in food processing. To the best of our knowledge, this is the first time for fluorescent sensing and imaging of CN^– in plant tissues. Moreover, our design also provides a new model scheme for the development of two-photon fluorescent nanomaterial, which is expected to hold great potential for food processing and safety testing