Portable Detection of Lysine Acetyltransferase Activity in Lung Cancer Cells Based on a Miniature Electrochemical Sensor.

The detection of lysine acetyltransferases is crucial for diagnosing and treating lung cancer, highlighting the necessity for highly efficient detection methods. We developed a portable, highly accurate, and sensitive technique using fast-scan cyclic voltammetry (FSCV) to determine the activity of the lysine acetyltransferase TIP60, employing a novel miniature electrochemical sensor. This approach involves a compact electrochemical cell, merely 3 mm in diameter, that holds solutions up to 50 µL, equipped with a conductive indium tin oxide working electrode. Uniquely, this system operates on a two-electrode model compatible with the FSCV, obviating the traditional requirement for a reference electrode. The system detects TIP60 activity through the continuous generation of CoA molecules that engage in reactions with Cu(II), thereby significantly improving the accuracy of the acetylation analysis. Remarkably, the detection limit achieved for TIP60 is notably low at 3.3 pg/mL (S/N = 3). The results show that the reversible dynamic acetylation can be effectively regulated by inhibitor incubation and glucose stimulation. This cutting-edge strategy enhances the analysis of a broad spectrum of biomarkers by modifying the responsive unit, and its miniaturization and portability significantly amplify its applicability in biomedical research, promising it to be a versatile tool for early diagnostic and therapeutic interventions in lung cancer.

Pt-Decorated Gold Nanoflares for High-Fidelity Phototheranostics: Reducing Side-Effects and Enhancing Cytotoxicity toward Target Cells.

Functionalized with the Au-S bond, gold nanoflares have emerged as promising candidates for theranostics. However, the presence of intracellular abundantly biothiols compromises the conventional Au-S bond, leading to the unintended release of cargoes and associated side-effects on non-target cells. Additionally, the hypoxic microenvironment in diseased regions limits treatment efficacy, especially in photodynamic therapy. To address these challenges, high-fidelity photodynamic nanoflares constructed on Pt-coated gold nanoparticles (Au@Pt PDNF) were communicated to avoid false-positive therapeutic signals and side-effects caused by biothiol perturbation. Compared with conventional photodynamic gold nanoflares (AuNP PDNF), the Au@Pt PDNF were selectively activated by cancer biomarkers and exhibited high-fidelity phototheranostics while reducing side-effects. Furthermore, the ultrathin Pt-shell catalysis was confirmed to generate oxygen which alleviated hypoxia-related photodynamic resistance and enhanced the antitumor effect. This design might open a new venue to advance theranostics performance and is adaptable to other theranostic nanomaterials by simply adding a Pt shell.

One-step, reagent-free construction of nano-enzyme as visual and reusable biosensor for oxidase substrates.

The substrates of oxidase are biologically essential substances that are closely associated with human physiological health. However, current biosensing methods suffer from tough recyclability and undesired denaturation of enzyme due to impurity interference. Herein, we have developed a visual and reusable biosensor for detecting substrate using glucose oxidase (GOx) as a model oxidase. GOx was immobilized onto gold nanoparticles (AuNPs) at -20 °C in one step without additional reagents. The resulting nano-enzyme generated coloimetric signals by coupling with horseradish peroxidase (HRP) using TMB as the substrate. Our results demonstrated that the immobilized GOx exhibited satisfactory sensitivity (0.68 µM) for glucose detection and higher inherent stability than free GOx under harsh conditions, enabling reliable detection of glucose in complex fluids (colored beverages and saliva). Furthermore, the nano-enzyme retained 80 % activity even after four cycles of catalytic oxidation. This strategy constructs a universal biosensor for substrates with nano-enzyme which rely only on intrinsic cysteine within the oxidase while avoiding functional handle modification.

Rigidity-Dependent Emission: Inspired Selection of an ATP-Specific Polyvalent Hydrogen Binding-Lighted Fluorophore for Intracellular Amplified Imaging.

ATP, a small molecule with high intracellular concentration (mM level), provides a fuel to power signal amplification, which is meaningful for biosensing. However, traditional ATP-powered amplification is based on ATP/aptamer recognition, which is susceptible to the complex biological microenvironment (e.g., nuclease). In this work, we communicate a signaling manner termed as ATP-specific polyvalent hydrogen binding (APHB), which is mimetic to ATP/aptamer binding but can avoid interference from biomolecules. The key in APHB is a functional fluorophore that can selectively bind with ATP via polyvalent hydrogen, and the fluorescence was lighted with the changes of the molecular structure from flexibility to rigidity. By designing, synthesizing, and screening a series of compounds, we successfully obtained an ATP-specific binding-lighted fluorophore (ABF). Experimental verification and a complex analogue demonstrated that two melamine brackets in the ABF dominate the polyvalent hydrogen binding between the ABF and ATP. Then, to achieve amplification biosensing, fibroblast activation protein (FAP) in activated hepatic stellate cells was taken as a model target, and a nanobeacon consisting of an ABF, a quencher, and an FAP-activated polymer shell was constructed. Benefiting from the ATP-powered amplification, the FAP was sensitively detected and imaged, and the potential relationship between differentiation of hepatocytes and FAP concentration was first revealed, highlighting the great potential of APHB-mediated signaling for intracellular sensing.

Brain-targeted Near-Infrared Nanobeacon for In Situ Monitoring H2S Fluctuation during Epileptic Seizures.

Epilepsy is a neurological brain disease, and its recurrent seizures are related to the reductive substance-powered antioxidant defense system (ADS). However, until now, there has been no report on the study of in situ antioxidant fluctuation during epilepsy of varying severity. In this work, hydrogen sulfide (H2S) was selected as the model target, a H2S-responsive near-infrared fluorophore was designed and synthesized, and an amphiphilic molecule was synthesized and modified with angiopep-2 peptide at its hydrophilic terminus. A nanobeacon termed as BFPP was prepared by the formation of micelles with the package of the fluorophore. The nanobeacon was sensitive to H2S, with a low detection limit of 17 nM. The H2S fluctuation in cells can be monitored by fluorescence imaging. In addition, angiopep-2 peptide at the surface of BFPP helps it cross the blood-brain barrier, and near-infrared fluorescence improves in vivo imaging. BFPP revealed that H2S was at a moderate level in the normal brain, but its level was obviously elevated during mild epilepsy because of the activation of the ADS while significantly suppressed during severe epilepsy due to neuronal damage. This approach is generally accessible for other targets by altering the responsive fluorophore, with significance for in situ analysis of brain pathology.

Bidirectional modulation of microRNA with a clamp-like triplex switch for enhanced and programmed gene therapy.

A clamp-like triplex switch (CTS) able to simultaneously downregulate an overexpressed onco-miRNA and replenish the lost tumor-suppressive miRNA in a controllable manner was developed for enhanced gene therapy. Compared to the "unidirectional" regulation approach, the CTS displayed improved anti-tumor efficacy in vitro and was harmless to healthy cells.

Zn2+ -Coordination-Driven RNA Assembly with Retained Integrity and Biological Functions.

Metal-coordination-directed biomolecule crosslinking in nature has been used for synthesizing various biopolymers, including DNA, peptides, proteins, and polysaccharides. However, the RNA biopolymer has been avoided so far, as due to the poor stability of the RNA molecules, the formation of a biopolymer may alter the biological function of the molecules. Herein, for the first time, we report Zn2+ -driven RNA self-assembly forming spherical nanoparticles while retaining the integrity and biological function of RNA. Various functional RNAs of different compositions, shapes, and lengths from 20 to nearly 1000 nucleotides were used, highlighting the versatility of this approach. The assembled nanospheres possess a superior RNA-loading efficiency, pharmacokinetics, and bioavailability. In-vitro and in-vivo evaluation demonstrated mRNA delivery for expressing GFP proteins, and microRNA delivery to triple-negative breast cancer. This coordination-directed self-assembly behavior amplifies the horizons of RNA coordination chemistry and the application scope of RNA-based therapeutics.

Thiol-suppressed I2-etching of AuNRs: acetylcholinesterase-mediated colorimetric detection of organophosphorus pesticides.

For the first time it is demonstrated that sulfhydryl compounds can suppress longitudinal etching of gold nanorods via consuming oxidizers, which provides a new signaling mechanism for colorimetric sensing. As a proof of concept, a colorimetric assay is developed for detecting organophosphorus pesticides, which are most widely used in modern agriculture to improve food production but with high toxicity to animals and the ecological environment. Triazophos was selected as a model organophosphorus pesticide. In the absence of triazophos, the active acetylcholinesterase can catalyze the conversion of acetylthiocholine iodide to thiocholine whose thiol group can suppress the I2-induced etching of gold nanorods. When triazophos is present, the activity of AchE is inhibited, and I2-induced etching of gold nanorods results in triazophos concentration-dependent color change from brown to blue, pink, and red. The aspect ratio of gold nanorods reduced with gradually blue-shifted longitudinal absorption. There was a linear detection range from 0 to 117 nM (R2 = 0.9908), the detection limit was 4.69 nM, and a good application potential was demonstrated by the assay of real water samples. This method will not only contribute to public monitoring of organophosphorus pesticides but also has verified a new signaling mechanism which will open up a new path to develop colorimetric detection methods. It has been first found that sulfhydryl compounds can suppress longitudinal etching of gold nanorods (AuNRs) via consuming oxidizers, which provides a new signaling mechanism for colorimetric sensing. As a proof of concept, a colorimetric assay is developed for sensitively detecting organophosphorus pesticides (OPs). It will not only contribute to public monitoring of OPs but also has verified a new signaling mechanism which will open up a new path to develop multicolor colorimetric methods.

Al centre-powered graphitic nanozyme with high catalytic efficiency for pH-independent chemodynamic therapy of cancer.

An Al centre-powered graphitic nanozyme derived from a metal organic framework was first developed for a chemodynamic tumor treatment. By virtue of the rapid and efficient generation of ˙OH in the slightly acidic tumor microenvironment, this nanozyme afforded high anti-tumor efficacy both in living cells and in vivo.

Pt-S Bond-Mediated Nanoflares for High-Fidelity Intracellular Applications by Avoiding Thiol Cleavage.

The Au-S bond is the classic way to functionalize gold nanoparticles (AuNPs). However, cleavage of the bond by biothiols and other chemicals is a long-standing problem hindering practical applications, especially in cells. Instead of replacing the thiol by a carbene or selenol for stronger adsorption, it is now shown that the Pt-S bond is much more stable, fully avoiding cleavage by biothiols. AuNPs were deposited with a thin layer of platinum, and an AuNP@Pt-S nanoflare was constructed to detect the miRNA-21 microRNA in living cells. This design retained the optical and cellular uptake properties of DNA-functionalized AuNPs, while showing high-fidelity signaling. It discriminated target cancer cells even in a mixed-cell culture system, where the Au-S based nanoflare was less sensitive. Compared to previous methods of changing the ligand chemistry, coating a Pt shell is more accessible, and previously developed methods for AuNPs can be directly adapted.

Human serum albumin as an intrinsic signal amplification amplifier for ultrasensitive assays of the prostate-specific antigen in human plasma.

As the most abundant protein in blood, human serum albumin (HSA) is usually regarded as an interferent in clinical molecular diagnosis. Herein, we report that HSA is an endogenous signal amplifier for the detection of the prostate-specific antigen (PSA) in human plasma. This is the first study to utilize intrinsic biological components as the signal amplifier in blood tests.

Visualization of Long Noncoding RNA MEG3 in Living Cells by a Triple-Helix-Powered 3D Catcher.

Visual imaging of long noncoding RNA (lncRNA) MEG3, a newfound regulator of transactivation and tumor growth suppression, is conducive to unlock the secrets of MEG3 in some important biological processes. Here, for the first time, we designed a DNA tetrahedron-based three-dimensional (3D) catcher for imaging cytoplasmic lncRNA MEG3 in living cells. The 3D catcher is composed of a triple-helix-forming dsDNA with capacity to bind the 5'-end GA-rich domain of the lncRNA MEG3 and four hairpin-shaped antisense sequences toward contiguous domain on MEG3. Once ingested by the cell, the 3D catcher quickly captures lncRNA MEG3 via forming a DNA-RNA triple-helix structure and triggering the hybridization-based string disassembly of the catcher. Concomitantly, the quenched hairpin is opened and the fluorescent signal undergoes lighting on conversion. Ascribed to the triple-helix-induced "domino effect," the disassembly reaction time is greatly shorter than the reaction with the inability to form a triple helix. The 3D catcher allows detection of long-chain targets as long as 129 nucleotide (129 nt) with a detection limit of 0.36 nM and distinguishes endogenous lncRNA MEG3 fragments in living cells between hepatoma cells and normal hepatocytes, which provides a reliable strategy for monitoring endogenous long fragment nucleic acid biomarkers in early clinical lesion diagnoses.

Cytoplasmic Protein-Powered In Situ Fluorescence Amplification for Intracellular Assay of Low-Abundance Analyte.

Fluorescence amplification is critical for in situ and real-time detection of intracellular low abundance biological species. However, current intracellular amplification techniques mainly rely on synthetic nucleic acid-based nanodevices, manipulating them in living cells remains challenging. To solve this problem, herein, a new signal amplification concept named cytoplasmic protein-powered in situ fluorescence amplification (CPFA) is proposed. CPFA takes cytoplasmic protein as cell-self-power for signal amplification enabling it to operate in living cells. To establish a prototype of CPFA, an amplifiable sensor for hydroxyl radicals (•OH) was designed by entrapping the screened cytoplasmic protein-enhanced fluorophore (PBF1) inside mesoporous silica (MSN) nanocontainer with ssDNA/PTAD-based signal switch. When encountered with •OH in living cells, the ssDNA was cleaved to separate PTAD from MSN, liberating multiple copies of the loaded PBF1 to light up the fluorescence. Furthermore, these released PBF1 molecules can instantly bind with cytoplasmic proteins to amplify their fluorescence signals. Take advantage of this two-stage amplification mode, the sensor in response to •OH exhibited remarkable fluorescence enhancement (near 400-fold) in cell lysates, and the •OH was linearly determined from 0 to 800 nM with a detection limit of 6.4 pM. Moreover, this sensor can track basal level and fluctuation of •OH in living cells on account of its high sensitivity. To our knowledge, this is the first effort to use cytoplasmic protein for amplifying detection signals, which will provide a new dimension to current methodologies for low-abundance biomarkers discovery and regulation for chemical biology and medical diagnostics.

MIL/Aptamer as a Nanosensor Capable of Resisting Nonspecific Displacement for ATP Imaging in Living Cells.

Fluorescent probes physisorbed on nanomaterials have emerged as a kind of useful and facile sensing platform for biological important molecules. However, nonspecific displacement in the physisorption systems is a non-negligible problem for the intracellular analysis. MIL (Materials of Institut Lavoisier), a subclass of metal-organic frameworks (MOFs), has high porosity, large surface area, and intriguing three-dimensional (3D) nanostructure with promising biological and biomedical applications such as molecular detection and drug delivery. Herein, we report MIL/aptamer-FAM as a nanosensor capable of resisting nonspecific displacement for intracellular adenosinetriphosphate (ATP) sensing and imaging. In this approach, by virtue of the remarkable quenching capability, high affinity of aptamers, and dramatic capability of resisting nonspecific displacement of 3D MIL-100, the assay and imaging of ATP in living cells were realized. Our results demonstrated that the MIL/aptamer-FAM nanosensor not only shows high selectivity for the detection of ATP in buffer but also is able to act as a "signal-on" nanosensor for specific imaging of ATP in living cells. The strategy reported here opens up a new way to develop MOF-based nanosensors for intracellular delivery and metabolite detection.

Real-Time Visualizing Mitophagy-Specific Viscosity Dynamic by Mitochondria-Anchored Molecular Rotor.

Mitophagy, as an evolutionarily conserved cellular process, plays a crucial role in preserving cellular metabolism and physiology. Various microenvironment alterations assigned to mitophagy including pH, polarity, and deregulated biomarkers are increasingly understood. However, mitophagy-specific viscosity dynamic in live cells remains a mystery and needs to be explored. Here, a water-soluble mitochondria-targetable molecular rotor, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9 H-carbazol-9-yl)] butanoate (BMVC), was exploited as a fluorescent viscosimeter for imaging viscosity variation during mitophagy. This probe contains two positively charged 1-methyl-4-vinylpyridium components as the rotors, whose rotation will be hindered with the increase of environmental viscosity, resulting in enhancement of fluorescence emission. The results demonstrated that this probe operates well in a mitochondrial microenvironment and displays an off-on fluorescence response to viscosity. By virtue of this probe, new discoveries such as the mitochondrial viscosity will increase during mitophagy are elaborated. The real-time visualization of the mitophagy process under nutrient starvation conditions was also proposed and actualized. We expect this probe would be a robust tool in the pathogenic mechanism research of mitochondrial diseases.

In†Situ Amplification-Based Imaging of RNA in Living Cells.

Owing to its important physiological functions, especially as molecular biomarkers of diseases, RNA is an important focus of biomedicine and biochemical sensing. Signal amplification detection has been put forward because of the need for accurate identification of RNA at low expression levels, which is significant for the early diagnosis and therapy of malignant diseases. However, conventional amplification methods for RNA analysis depend on the use of enzymes, fixation of cells, and thermal cycling, which confine their performance to cell lysates or dead cells, thus the imaging of RNA in living cells remained until recently little explored. In recent years, the advance of isothermal amplification of nucleic acids has opened paths for meeting this need in living cells. This minireview tracks the development of in situ amplification assays for RNAs in living cells, and highlights the potential challenges facing this field, aiming to improve the development of in vivo isothermal amplification as well as usher in new frontiers in this fertile research area.

A Ratiometric Two-Photon Fluorescent Cysteine Probe with Well-Resolved Dual Emissions Based on Intramolecular Charge Transfer-Mediated Two-Photon-FRET Integration Mechanism.

The development of an efficient ratiometric two-photon fluorescence imaging probe is crucial for in situ monitoring of biothiol cysteine (Cys) in biosystems, but the current reported intramolecular charge transfer (ICT)-based one suffers from serious overlap between the shifted emission bands. To address this issue, we herein for the first time constructed an ICT-mediated two-photon excited fluorescence resonance energy transfer (TP-FRET) system consisting of a two-photon fluorogen benzo[ h]chromene and a Cys-responsive benzoxadiazole-analogue dye. Different from a previous mechanism that utilized single two-photon fluorogen to acquire a ratiometric signal, ICT was used to switch on the TP-FRET process of the energy transfer dyad by eliciting an absorption shift of benzoxadiazole with Cys to modulate the spectral overlap level between benzo[ h]chromene emission and benzoxadiazole absorption, resulting in two well-separated emission signal changes with large emission wavelength shift (120 nm), fixed two-photon excitation maximum (750 nm), and significant variation in fluorescence ratio (over 36-fold). Therefore, it can be successfully employed to ratiometrically visualize Cys in HeLa cells and liver tissues. Importantly, this new ICT-mediated TP-FRET integration mechanism would be convenient for designing ratiometric two-photon fluorescent probes with two well-resolved emission spectra suitable for high resolution two-photon fluorescence bioimaging.

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.

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.

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.