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.

Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

Lowering Entropic Barriers in Triplex DNA Switches Facilitating Biomedical Applications at Physiological pH.

Triplex DNA switches are attractive allosteric tools for engineering smart nanodevices, but their poor triplex-forming capacity at physiological conditions limited the practical applications. To address this challenge, we proposed a low-entropy barrier design to facilitate triplex formation by introducing a hairpin duplex linker into the triplex motif, and the resulting triplex switch was termed as CTNSds. Compared to the conventional clamp-like triplex switch, CTNSds increased the triplex-forming ratio from 30 % to 91 % at pH 7.4 and stabilized the triple-helix structure in FBS and cell lysate. CTNSds was also less sensitive to free-energy disturbances, such as lengthening linkers or mismatches in the triple-helix stem. The CTNSds design was utilized to reversibly isolate CTCs from whole blood, achieving high capture efficiencies (>86 %) at pH 7.4 and release efficiencies (>80 %) at pH 8.0. Our approach broadens the potential applications of DNA switches-based switchable nanodevices, showing great promise in biosensing and biomedicine.

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.

A Reversible Fluorescent Probe for In Situ Monitoring Redox Imbalance during Mitophagy.

Mitophagy is the lysosome-dependent degradation of damaged and dysfunctional mitochondria, which is closely associated with H2O2-related redox imbalance and pathological processes. However, development of fast-responding and highly sensitive reversible fluorescent probes for monitoring of mitochondrial H2O2 dynamics is still lacking. Herein, we report a reversible fluorescent probe (M-HP) that enables real-time imaging of H2O2-related redox imbalance. In vitro studies demonstrated that M-HP had a rapid response and high sensitivity to the H2O2/GSH redox cycle, with a detection limit of 17 nM for H2O2. M-HP was applied to imaging of H2O2 fluctuation in living cells with excellent reversibility and mitochondrial targeting. M-HP reveals an increase in mitochondrial H2O2 under lipopolysaccharide stimulation and a decrease in H2O2 following the combined treatment with rapamycin. This suggests that the level of oxidative stress is significantly suppressed after the enhancement of mitophagy. The rationally designed M-HP offers a powerful tool for understanding redox imbalance during mitophagy.

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.

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.

Platinum Nanoparticles Loaded Graphitic Carbon Nitride Nanosheets with Enhanced Peroxidase-like Activity for H2O2 and Oxidase-Based Sensing.

Platinum nanoparticles (PtNPs) are classical peroxidase-like nanozyme; self-agglomeration of nanoparticles leads to the undesirable reduction in stability and catalytic activity. Herein, a hybrid peroxidase-like nanocatalyst consisting of PtNPs in situ growing on g-C3N4 nanosheets with enhanced peroxidase-mimic catalytic activity (PtNP@g-C3N4 nanosheets) was prepared for H2O2 and oxidase-based colorimetric assay. g-C3N4 nanosheets can be used as carriers to solve the problem of poor stability of PtNPs. We observed that the catalytic ability could be maintained for more than 90 days. PtNP@g-C3N4 nanosheets could quickly catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), and the absorbance of blue color oxidized TMB (oxTMB) showed a robust linear relationship with the concentration of H2O2 (the detection limit (LOD): 3.33 µM). By utilizing H2O2 as a mediator, this strategy can be applied to oxidase-based biomolecules (glucose, organophosphorus, and so on, that generate or consume hydrogen peroxide) sensing. As a proof of concept, a sensitive assay of cholesterol that combined PtNP@g-C3N4 nanosheets with cholesterol oxidase (ChOx) cascade catalytic reaction was constructed with an LOD of 9.35 µM in a widespread range from 10 to 800 µM (R2 = 0.9981). In addition, we also verified its ability to detect cholesterol in fetal bovine serum. These results showed application prospect of PtNP@g-C3N4 nanosheets-based colorimetry in sensing and clinical medical detection.

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.

A Polymeric Nanobeacon for Monitoring the Fluctuation of Hydrogen Polysulfides during Fertilization and Embryonic Development.

Fertilization and early embryonic development as the beginning of a new life are key biological events. Hydrogen polysulfide (H2 Sn ) plays important roles during physiological regulation, such as antioxidation-protection. However, no report has studied in situ H2 Sn fluctuation during early embryonic development because of the low abundance of H2 Sn and inadequate sensitivity of probes. We herein construct a polymeric nanobeacon from a H2 Sn -responsive polymer and fluorophores, which is capable of detecting H2 Sn selectively and of signal amplification. Taking the zebrafish as a model, the polymeric nanobeacon revealed that the H2 Sn level was significantly elevated after fertilization due to the activation of cell multiplication, suppressed partially during embryonic development, and finally kept steady up to zebrafish emergence. This strategy is generally accessible for biomarkers by altering the responsive unit and significant for facilitating biological analysis during life development.

Self-Immolative Dye-Doped Polymeric Probe for Precisely Imaging Hydroxyl Radicals by Avoiding Leakage.

For sensing low abundance of biomarkers, utilizing nanocarriers to load dyes is an efficient method to amplify the detected signal. However, the non-specific leak of the internal dyes in this approach is accompanied by false positive signals, resulting in inaccurate signal acquirement. To address this issue, in this work, we reported a novel signal amplification strategy with dye as a scaffold to construct a self-immolative dye-doped polymeric probe (SDPP). In our proposed approach, the dyes were covalently integrated into the main chain of a polymer, which can avoid the non-specific leak of the dye when used in a rigorous biological environment, thus evading the false positive signal. As a prototype of this concept, a SDPP, which responds to hydroxyl radicals (•OH), was rationally fabricated. Upon being activated by •OH, SDPP will liberate the dye through a self-immolative reaction to bind with protein for amplifying the fluorescence signal. Compared with a dye-loaded nanoprobe, SDPP can precisely track intracellular basal •OH levels and visualize the •OH associated with myocarditis in vivo. More importantly, the attempt in this work not only provides an effective molecular tool to investigate the role of •OH in cardiopathy, but also puts forward a new direction to current signal-amplifying strategies for precisely and reliably acquiring the intracellular molecular information.

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.

A Glucose-Powered Activatable Nanozyme Breaking pH and H2 O2 Limitations for Treating Diabetic Infections.

The peroxidase-like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2 O2 into . OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2 O2 . We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2 O2 limitations under physiological conditions. As a proof of concept, aptamer-functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule "APGH", which can be activated by bacteria-secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2 O2 for the in-situ generation of . OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.

Human Serum Albumin-Occupying-Based Fluorescence Turn-On Analysis of Antiepileptic Drug Tiagabine Hydrochloride.

Tiagabine hydrochloride (TGB) is a clinically frequently used drug for anticonvulsion and reducing epileptic frequency. Over administration of TGB could bring about adverse effects, such as speech disorder, depression, and even suicidal tendencies. Therefore, accessible and sensitive assay for analysis of TGB becomes an urgent need toward guiding clinical medication. Here, we present the first report on fluorescence turn-on detection of TGB in urine testing. In this protocol, a fluorescent dye, perylene tetracarboxylic acid imide derivative (PTAI), is found specifically occupying the Sudlow site II of human serum albumin (HSA) and displays a new phenomenon of binding-induced quenching (BIQ). In presence of TGB, competitive binding of the TGB to the site II of HSA will trigger release of PTAI, thus successfully lighting up the fluorescence of PTAI. This label-free assay enjoys a broader working range (1-350 µM) and lower detection limit (0.218 µM) than the traditional liquid chromatography method and is uninterfered by the miscellaneous in the artificial urine. The BIQ probe highlights the merits of HSA as a quencher and a molecular recognition unit, and it opens up a way for studying drug-HSA interaction mechanism and noninvasive pharmaceutical testing.

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.

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.

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.

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.

Colorimetric aminotriazole assay based on catalase deactivation-dependent longitudinal etching of gold nanorods.

A colorimetric and visual assay is described for the herbicide aminotriazole (ATZ). It is based on the etching of gold nanorods (AuNRs) by iodine which is formed on oxidation of iodide via H2O2. Longitudinal etching of the AuNRs occurs quickly and is accompanied by a color change from dark blue to red. In the absence of ATZ and the presence of active catalase (CAT), H2O2 is quickly decomposed into water, and the AuNRs will not be etched. In the presence of ATZ, CAT is partially deactivated, and this affects the amount of available H2O2 and, consequently, of the iodine. Hence, the color is significantly changed. The color changes can be easily detected with bare eyes. The assay has a linear response in the 5 to 70 µM concentration range, with a detection limit of 1.3 µM and high selectivity for ATZ. It was applied to the determination of ATZ in water and food samples. Graphical abstract A multicolor colorimetric method is developed for aminotriazole (ATZ) detection based on catalase (CAT) deactivation-dependent longitudinal etching of gold nanorods (AuNRs). The color signals can be visually identified. Good detection performances and capability for evaluating ATZ level in water and food samples is demonstrated.