Click Conjugation of Zwitterionic Peptide with DNA Strand: An Efficient Antifouling Strategy for Versatile Photoelectrochemical Aptasensor.

Advanced antifouling biosensors have garnered considerable attention for their potential for precise and sensitive analysis in complex human bodily fluids. Herein, a pioneering approach was utilized to establish a robust and versatile photoelectrochemical aptasensor by conjugating a zwitterionic peptide with a DNA strand. Specifically, the branched zwitterionic peptide (BZP) was efficiently linked to complementary DNA (cDNA) through a click reaction, forming the BZP-cDNA conjugate. This intriguing conjugate exploited the BZP domain to create an antifouling biointerface, while the cDNA component facilitated subsequent hybridization with probe DNA (pDNA). To advance the development of the aptasensor, an upgraded PDA/HOF-101/ZnO ternary photoelectrode was designed as the signal converter for the modification of the BZP-cDNA conjugate, while a bipyridinium (MCEPy) molecule with strong electron-withdrawing properties was labeled at the front end of the pDNA to form the pDNA-MCEPy signal probe. Targeting the model of mucin-1, a remarkable enhancement in the photocurrent signal was achieved through exonuclease-I-aided target recycling. Such an engineered zwitterionic peptide-DNA conjugate surpasses the limitations imposed by conventional peptide-based sensing modes, exhibiting unique advantages such as versatility in design and capability for signal amplification.

Intelligent Cell Profiling and Precision Release: Multimolecular Marker-Activated Transmembrane DNA Computing Nanosystem.

Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.

Engineered Branching Peptide as Dual-Functional Antifouling and Recognition Probe: Toward a Dual-Photoelectrode Protein Biosensor with High Accuracy.

The building of practical biosensors that have anti-interference abilities against biofouling of nonspecific proteins and biooxidation of reducing agents in actual biological matrixes remains a great challenge. Herein, a robust photoelectrochemical (PEC) biosensor capable of accurate detection in human serum was pioneered through the integration of a new engineered branching peptide (EBP) into a synergetic dual-photoelectrode system. The synergetic dual-photoelectrode system involved the tandem connection of a C3N4/TiO2 photoanode and a AuPt/PANI photocathode, while the EBP as a dual-functional antifouling and recognition probe featured an inverted Y-shaped configuration with one recognition backbone and two antifouling branches. Such an EBP enables a simple procedure for electrode modification and an enhanced antifouling nature compared to a regular linear peptide (LP), as theoretically supported by the results from molecular dynamics simulations. The as-developed PEC biosensor had a higher photocurrent response and a good antioxidation property inherited from the photoanode and photocathode, respectively. Targeting the model protein biomarker of cardiac troponin I (cTnI), this biosensor achieved good performances in terms of high sensitivity, specificity, and anti-interference.

Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a.

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging.

Accurate identification of cancer cells is an essential prerequisite for cancer diagnosis and subsequent effective curative interventions. The logic-gate-assisted cancer imaging system that allows a comparison of expression levels between biomarkers, rather than just reading biomarkers as inputs, returns a more comprehensive logical output, improving its accuracy for cell identification. To fulfill this key criterion, we develop a compute-and-release logic-gated double-amplified DNA cascade circuit. This novel system, CAR-CHA-HCR, consists of a compute-and-release (CAR) logic gate, a double-amplified DNA cascade circuit (termed CHA-HCR), and a MnO2 nanocarrier. CAR-CHA-HCR, a novel adaptive logic system, is designed to logically output the fluorescence signals after computing the expression levels of intracellular miR-21 and miR-892b. Only when miR-21 is present and its expression level is above the threshold CmiR-21 > CmiR-892b, the CAR-CHA-HCR circuit performs a compute-and-release operation on free miR-21, thereby outputting enhanced fluorescence signals to accurately image positive cells. It is capable of comparing the relative concentrations of two biomarkers while sensing them, thus allowing accurate identification of positive cancer cells, even in mixed cell populations. Such an intelligent system provides an avenue for highly accurate cancer imaging and is potentially envisioned to perform more complex tasks in biomedical studies.

Engineering of a novel D-A type fluorophore with hydrogen bond-induced enhanced emission property for sensitively detecting endogenous HOCl in living cells and tissues.

Fluorescence imaging has been widely employed for biomedical research and clinical diagnostics. With ease of synthesis and excellent photophysical properties, D-A type fluorophores are widely designed for fluorescence imaging. However, traditional D-A type fluorophores are solvatochromic which reduces the fluorescence brightness in the biological system. To solve this problem and build on our previous work, we devised a novel HIEE fluorophore MTC with typical anti-solvatochromic fluorescence. Furthermore, the activated fluorescent probe designed based on MTC showed excellent imaging performance. We believe that the strategy based on the fluorophores with typical anti-solvatohromic fluorescence can be a useful platform for designing fluorescent probes for high-brightness imaging in the biological system.

Molecular Engineering of Near-Infrared Fluorescent Probes for Cell Membrane Imaging.

Cell membrane (CM) is a phospholipid bilayer that maintains integrity of a whole cell and relates to many physiological and pathological processes. Developing CM imaging tools is a feasible method for visualizing membrane-related events. In recent decades, small-molecular fluorescent probes in the near-infrared (NIR) region have been pursued extensively for CM staining to investigate its functions and related events. In this review, we summarize development of such probes from the aspect of design principles, CM-targeting mechanisms and biological applications. Moreover, at the end of this review, the challenges and future research directions in designing NIR CM-targeting probes are discussed. This review indicates that more efforts are required to design activatable NIR CM-targeting probes, easily prepared and biocompatible probes with long retention time regarding CM, super-resolution imaging probes for monitoring CM nanoscale organization and multifunctional probes with imaging and phototherapy effects.

A sensitive fluorescence biosensor based on metal ion-mediated DNAzyme activity for amplified detection of acetylcholinesterase.

In this paper, we developed an amplified fluorescence biosensor for acetylcholinesterase (AChE) activity detection by taking advantage of the mercury ion-mediated Mgzyme (Mg2+-dependent DNAzyme) activity. The catalytic activity of Mgzyme can be inhibited by the formation of T-Hg2+-T base pairs between the Mgzyme and mercury ions. Therefore, the Mgzyme-Hg2+ complex has no activity on a molecular beacon (MB) substrate, which afforded a very weak fluorescence background for this biosensor. After the addition of acetylcholinesterase (AChE), the substrate acetylthiocholine could be hydrolyzed to thiocholine, which has a stronger binding power with mercury ions than T-Hg2+-T base pairs. Therefore, the Mgzyme activity was recovered. The activated Mgzyme could hybridize with the MB substrate and undergo many cleavage cycles, resulting in a significant increase of fluorescence intensity. This biosensor displayed high sensitivity with the detection limit as low as 0.01 mU mL-1. Moreover, this design did not require complex composition and sequence design; thus it is simple and convenient. This biosensor was also applied for the determination of AChE in human blood and showed satisfactory results.

Long-wavelength emission carbon dots as self-ratiometric fluorescent nanoprobe for sensitive determination of Zn2.

A novel ratiometric fluorescence nanoprobe based on long-wavelength emission carbon dots (CDs) was designed for high sensitive and selective detection of Zn2+. The CDs were conveniently prepared by a one-step solvothermal treatment of formamide and glutathione (GSH). Under single excitation wavelength (420 nm), the obtained CDs exhibit three emission peaks at 470, 650, and 685 nm, respectively. For the long-wavelength emission region of the CDs, the fluorescence at 685 nm can be quenched with different levels upon the addition of most metal ions. However, the presence of Zn2+ not only results in the fluorescence quenching at 685 nm effectively but also enhances at 650 nm remarkably, which may be due to the formation of CD-Zn2+ chelate complex inducing the dispersion of CDs aggregates and changes in the group distribution on the surface of CDs. Taking the advantage of the unique fluorescence response induced by Zn2+, the prepared CDs were successfully employed as nanoprobe for self-ratiometric fluorescence determination of Zn2+ with F650/F685 as signal output. A good linear relationship in the concentration range 0.01 to 2 µM, and a detection limit as low as 5.1 nM has been obtained. The ratiometric nanoprobe was successfully applied to  Zn2+ determination  in human serum samples.

Ratiometric Semiconducting Polymer Nanoparticle for Reliable Photoacoustic Imaging of Pneumonia-Induced Vulnerable Atherosclerotic Plaque in Vivo.

Acute pneumonia can greatly increase the vulnerable risk of atherosclerotic plaque and contribute to the mortality of cardiovascular disease. To accurately assess the rupture risk caused by acute pneumonia, we developed a novel kind of ratiometric semiconducting polymer nanoparticle (RSPN) for photoacoustic imaging of vulnerable plaque in apolipoprotein E-deficient mice complicated with pneumonia. Specifically, RSPN can react with O2•- and exhibit the enhanced photoacoustic signals at about 690 nm, while 800 nm is regarded as an internal photoacoustic reference. As a result, RSPN can provide reliable determination of O2•- within aortic atherosclerosis by analyzing the ratios of photoacoustic signals, which can successfully reflect the oxidative stress level in vulnerable plaque. Therefore, RSPN enable to specifically distinguish plaque-bearing mice and plaque-bearing mice complicated with pneumonia from healthy mice, which provides a promising tool to predict the vulnerability of plaque for reducing the mortality of atherosclerotic-induced cardiovascular disease.

Covalent Amide-Bonded Nanoflares for High-Fidelity Intracellular Sensing and Targeted Therapy: A Superstable Nanosystem Free of Nonspecific Interferences.

A nanoflare, a conjugate of Au nanoparticles (NPs) and fluorescent nucleic acids, is believed to be a powerful nanoplatform for diagnosis and therapy. However, it highly suffers from the nonspecific detachment of nucleic acids from the AuNP surface because of the poor stability of Au-S linkages, thereby leading to the false-positive signal and serious side effects. To address these challenges, we report the use of covalent amide linkage and functional Au@graphene (AuG) NP to fabricate a covalent conjugate system of DNA and AuG NP, label-rcDNA-AuG. Covalent coating of abundant amino groups (-NH2) onto the graphitic shell of AuG NP efficiently facilitates the coupling with carboxyl-labeled capture DNA sequences through simple, but strong, amide bonds. Importantly, such an amide-bonded nanoflare possesses excellent stability and anti-interference capability against the biological agents (nuclease, DNA, glutathione (GSH), etc.). By accurately monitoring the intracellular miR-21 levels, this covalent nanoflare is able to identify the positive cancer cells even in a mix of cancer and normal cells. Moreover, it allows for efficient photodynamic therapy of the targeted cancer cells with minimized side effects on normal cells. This work provides a facile approach to develop a superstable nanosystem showing promising potential in clinical diagnostics and therapy.

Tumor-Specific Multipath Nucleic Acid Damages Strategy by Symbiosed Nanozyme@Enzyme with Synergistic Self-Cyclic Catalysis.

The high proliferation efficiency, redox imbalance, and elevated nucleic acid repair capabilities of tumor cells severely restrict the theranostic efficacy. Selectively interference chaotic tumors with devastating nucleic acid damages (NUDs) properties are expected to overcome theranostic barriers. Here, an exquisite catalytic-based strategy with comprehensive NUDs mechanisms is demonstrated. In this regard, enzyme (glucose oxidase, GOD) symbioses nanozyme Cu3+x (PO4 )2 through biomineralization (abbreviated as Cu@GOD), GOD can disorder the metabolism by consuming glucose, thereby inhibiting the nutrition supply for nucleic acid repair. GOD-catalyzed H2 O2 guarantees the self-cyclic glutathione depletion and reactive oxygen species generation caused by Cu3+x (PO4 )2 , resulted the reduced antioxidation defense and enhanced oxidation assault, ensures an indiscriminate NUDs ability. Moreover, the high photothermal effect of Cu3+x (PO4 )2 induces effective tumor inhibition. Consequently, this substantial multipath NUDs strategy, with potentials of suppressing the cytoprotective mechanisms, amplifying the cellular oxidative stress, and disrupting the redox balance to ensure substantial irreversible NUDs, completely breaks the obstacle of chaotic tumors, providing new conceptual thinking for tumor proliferation inhibition.

Discovery of new 2-phenyl-1H-benzo[d]imidazole core-based potent α-glucosidase inhibitors: Synthesis, kinetic study, molecular docking, and in vivo anti-hyperglycemic evaluation.

In the present study, a series of 2-phenyl-1H-benzo[d]imidazole-based α-glucosidase inhibitors were synthesized and evaluated for their in vitro and in vivo anti-diabetic potential. Screening of an in-house library revealed a moderated α-glucosidase inhibitor, 6a with 3-(1H-benzo[d]imidazol-2-yl)aniline core, and then the structural optimization was performed to obtain more efficient derivatives. Most of these derivatives showed increased activity than 6a, and the most promising inhibitors were found to be compounds 15o and 22d with IC50 values of 2.09 ± 0.04 and 0.71 ± 0.02 µM, respectively. Fluorescence quenching experiment confirmed the direct binding of compounds 15o and 22d with α-glucosidase. Kinetic study revealed that both compounds were non-competitive inhibitors, that was consistent with the result of molecular docking studies where they located at the allosteric site of the enzyme. Cell viability evaluation demonstrated the non-cytotoxicity of 15o and 22d against LO2 cells. Furthermore, the in vivo pharmacodynamic study revealed that compound 15o showed significant hypoglycemic activity and improved oral sucrose tolerance, comparable to the positive control acarbose.

A novel ratiometric fluorescence nanoprobe for sensitive determination of uric acid based on CD@ZIF-CuNC nanocomposites.

A novel ratiometric fluorescence nanoprobe based on carbon dots (CDs) and Cu nanoclusters (CuNCs) was designed for the label-free determination of uric acid (UA). The metal-organic framework (MOF) encapsulated CuNCs (ZIF-CuNC), and nitrogen-doped CDs can self-assemble into well-defined spherical nanocomposites (CD@ZIF-CuNC) due to physical adsorption. Under the excitation wavelength of 360 nm, the CD@ZIF-CuNC nanocomposites exhibit two evident intrinsic emissions peaked at 460 nm (CDs) and 620 nm (ZIF-CuNC), respectively. In the presence of H2O2, the fluorescence of CD@ZIF-CuNC at 620 nm is quenched remarkably within 1 min, while little effect on the emission at 460 nm is observed. Therefore, taking the fluorescence at 620 nm as the report signal and 460 nm as the reference signal, ratiometric quantitative determination of H2O2 was achieved with a linear range of 1-100 µM and a detection limit of 0.30 µM. The CD@ZIF-CuNC nanoprobe was successfully applied to the determination of UA that is catalyzed by uricase to produce H2O2, obtaining the linear range of 1-30 µM and the detection limit of 0.33 µM. Eventually, this strategy has been successfully applied to the determination of UA in human urine samples. A novel and convenient CDs@ZIF-CuNCs-based nanoplatform was constructed for sensitive ratiometric fluorescence determination of UA.

Shell-Switchable SERS Blocking Strategy for Reliable Signal-On SERS Sensing in Living Cells: Detecting an External Target without Affecting the Internal Raman Molecule.

For SERS analysis in living cells, the inevitable desorption of Raman molecule on the substrate surface is a key challenge. To ensure high stability, SERS systems with Raman molecules protected inside the core-Raman molecule-shell (C-M-S) structures have been designed, but at the expense of sacrificed sensing performances. Here a shell-switchable SERS blocking strategy is developed for the reliable SERS analysis in living cells, relying on the shell blockers to regulate the SERS sensing signal without affecting the internal Raman molecules. After several C-M-S structures were investigated, the SERS blocking mechanism confirmed that thick shells (Au, Ag, ZnO, and MnO2) can cause a significant reduction in the internal SERS signal by obstructing the penetration of the laser or signal. The CAu-Mpy-SAu-SMnO2 nanoprobe is designed for the ratiometric SERS sensing in living cells, which retains sensing performances even though the Raman molecule is protected inside the nanostructure. This SERS strategy makes the turn-on sensing achievable in living cells with the MnO2 shell as a signal switch and a Raman reference. Additionally, it allows for accurate monitoring of the degradation of MnO2 carriers in living cells, even without fluorescent labels.

Biodegradable nanoprobe based on MnO2 nanoflowers and graphene quantum dots for near infrared fluorescence imaging of glutathione in living cells.

Near infrared (NIR) emitting semiconductor quantum dots can be excellent fluorescent nanoprobes, but the poor biodegradability and potential toxicity limits their application. The authors describe a fluorescent system composed of graphene quantum dots (GQDs) as NIR emitters, and novel MnO2 nanoflowers as the fluorescence quenchers. The system is shown to be an activatable and biodegradable fluorescent nanoprobe for the "turn-on" detection of intracellular glutathione (GSH). The MnO2-GQDs nanoprobe is obtained by adsorbing GQDs onto the surface of MnO2 nanoflowers through electrostatic interaction. This results in the quenching of the NIR fluorescence of the GQDs. In the presence of GSH, the MnO2-GQDs nanoprobe is degraded and releases Mn2+ and free GQDs, respectively. This gives rise to increased fluorescence. The nanoprobe displays high sensitivity to GSH and with a 2.8 µM detection limit. It integrates the advantages of NIR fluorescence and biodegradability, selectivity, biocompatibility and membrane permeability. All this makes it a promising fluorescent nanoprobe for GSH and for cellular imaging of GSH as shown here for the case of MCF-7 cancer cells. Graphical abstract A biodegradable NIR fluorescence nanoprobe (MnO2-GQDs) for the "turn-on" detection of GSH in living cell was established, with the NIR GQD as the fluorescence reporter and the MnO2 nanoflower as the fluorescence quencher.

Near Infrared Graphene Quantum Dots-Based Two-Photon Nanoprobe for Direct Bioimaging of Endogenous Ascorbic Acid in Living Cells.

Ascorbic acid (AA), as one of the most important vitamins, participates in various physiological reactions in the human body and is implicated with many diseases. Therefore, the development of effective methods for monitoring the AA level in living systems is of great significance. Up to date, various technologies have been developed for the detection of AA. However, few methods can realize the direct detection of endogenous AA in living cells. In this work, we for the first time reported that near-infrared (NIR) graphene quantum dots (GQD) possessed good two-photon fluorescence properties with a NIR emission at 660 nm upon exciting with 810 nm femtosecond pulses and a two-photon (TP) excitation action cross-section (δΦ) of 25.12 GM. They were then employed to construct a TP nanoprobe for detection and bioimaging of endogenous AA in living cells. In this nanosystem, NIR GQDs (NGs), which exhibited lower fluorescence background in living system to afford improved fluorescence imaging resolution, were acted as fluorescence reporters. Also CoOOH nanoflakes were chosen as fluorescence quenchers by forming on the surface of NGs. Once AA was introduced, CoOOH was reduced to Co2+, which resulted in a "turn-on" fluorescence signal of NGs. The proposed nanoprobe demonstrated high sensitivity toward AA, with the observed LOD of 270 nM. It also showed high selectivity to AA with excellent photostability. Moreover, the nanoprobe was successfully used for TP imaging of endogenous AA in living cells as well as deep tissue imaging.

Quench-Shield Ratiometric Upconversion Luminescence Nanoplatform for Biosensing.

Upconversion nanoparticles (UCNPs) possess several unique features, but they suffer from surface quenching effects caused by the interaction between the UCNPs and fluorophore. Thus, the use of UCNPs for target-induced emission changes for biosensing and bioimaging has been challenging. In this work, fluorophore and UCNPs are effectively separated by a silica transition layer with a thickness of about 4 nm to diminish the surface quenching effect of the UCNPs, allowing a universal and efficient luminescence resonance energy transfer (LRET) ratiometric upconversion luminescence nanoplatform for biosensing applications. A pH-sensitive fluorescein derivative and Hg(2+)-sensitive rhodamine B were chosen as fluoroionphores to construct the LRET nanoprobes. Both showed satisfactory target-triggered ratiometric upconversion luminescence responses in both solution and live cells, indicating that this strategy may find wide applications in the design of nanoprobes for various biorelated targets.

Alkyne-functionalized superstable graphitic silver nanoparticles for Raman imaging.

Noble metals, especially gold, have been widely used in plasmon resonance applications. Although silver has a larger optical cross section and lower cost than gold, it has attracted much less attention because of its easy corrosion, thereby degrading plasmonic signals and limiting its applications. To circumvent this problem, we report the facile synthesis of superstable AgCu@graphene (ACG) nanoparticles (NPs). The growth of several layers of graphene onto the surface of AgCu alloy NPs effectively protects the Ag surface from contamination, even in the presence of hydrogen peroxide, hydrogen sulfide, and nitric acid. The ACG NPs have been utilized to enhance the unique Raman signals from the graphitic shell, making ACG an ideal candidate for cell labeling, rapid Raman imaging, and SERS detection. ACG is further functionalized with alkyne-polyethylene glycol, which has strong Raman vibrations in the Raman-silent region of the cell, leading to more accurate colocalization inside cells. In sum, this work provides a simple approach to fabricate corrosion-resistant, water-soluble, and graphene-protected AgCu NPs having a strong surface plasmon resonance effect suitable for sensing and imaging.

Magnetic graphitic nanocapsules for programmed DNA fishing and detection.

Graphene nanomaterials are typically used in biosensing applications, and they have been demonstrated as good fluorescence quenchers. While many conventional amplification platforms are available, developing new nanomaterials and establishing simple, enzyme-free and low-cost strategies for high sensitivity biosensing is still challenging. Therefore, in this work, a core-shell magnetic graphitic nanocapsule (MGN) material is synthesized and its capabilities for the detection of biomolecules are investigated. MGN combines the unique properties of graphene and magnetic particles into one simple and sensitive biosensing platform, which quenches around 98% of the dye fluorescence within minutes. Based on a programmed multipurpose DNA capturing and releasing strategy, the MGN sensing platform demonstrates an outstanding capacity to fish, enrich, and detect DNA. Target DNA molecules as low as 50 pM could be detected, which is 3-fold lower than the limit of detection commonly achieved by carbon nanotube and graphene-based fluorescent biosensors. Moreover, the MGN platform exhibits good sensing specificity against DNA mismatch tests. Overall, therefore, these magnetic graphitic nanocapsules demonstrate a promising tool for molecular disease diagnosis and biomedicine. This simple fishing and enrichment strategy may also be extended to other biological and environmental applications and systems.