Simultaneous tracking of autophagy and oxidative stress during stroke with an ICT-TBET integrated ratiometric two-photon platform

Over recent years, fluorescent probes exhibiting simultaneous responses to multiple targets have been developed for in situ, real-time monitoring of cellular metabolism using two photon fluorescence sensing techniques due to numerous advantages including ease of operation, rapid reporting, high resolution, long visualization time and being non-invasive. However, due to interference from different fluorescence channels during simultaneous monitoring of multiple targets and the lack of ratiometric capability amongst the available probes, the accuracy in tracing metabolic processes has been restricted. With this research, using a through-bond energy transfer (TBET) mechanism, we designed a viscosity and peroxynitrite (ONOO⁻) mitochondria-targeting two-photon ratiometric fluorescent probe Mito-ONOO. Our results indicated that with decreasing levels of mitochondrial viscosity and increasing levels of ONOO⁻, the maximum of the emission wavelength of the probe shifted from 621 nm to 495 nm under 810 nm two-photon excitation. The baselines for the two emission peaks were significantly separated (Δλ = 126 nm), improving the resolution and reliability of bioimaging. Moreover, by ratiometric analysis during oxygen-glucose deprivation/reoxygenation (OGD/R, commonly used to simulate cell ischemia/reperfusion injury), the real-time visualization of the metabolic processes of autophagy and oxidative stress was possible. Our research indicated that during cellular oxygen-glucose deprivation/reoxygenation, cells produce ONOO⁻, causing cellular oxidative stress and cellular autophagy after 15 min, as such Mito-ONOO exhibits the potential for the monitoring and diagnosis of stroke, as well as providing insight into potential treatments, and drug design.

Ratiometric simultaneous tracking of autophagy and oxidative stress was achieved using an ICT-TBET integrated platform. Mito-ONOO exhibited excellent selectivity, good chemical stability, and non-overlapping ratiometric signals.     

Enzyme-activatable fluorescent probes for β-galactosidase: from design to biological applications

β-Galactosidase (β-gal), a typical hydrolytic enzyme, is a vital biomarker for cell senescence and primary ovarian cancers. Developing precise and rapid methods to monitor β-gal activity is crucial for early cancer diagnoses and biological research. Over the past decade, activatable optical probes have become a powerful tool for real-time tracking and in vivo visualization with high sensitivity and specificity. In this review, we summarize the latest advances in the design of β-gal-activatable probes via spectral characteristics and responsiveness regulation for biological applications, and particularly focus on the molecular design strategy from turn-on mode to ratiometric mode, from aggregation-caused quenching (ACQ) probes to aggregation-induced emission (AIE)-active probes, from near-infrared-I (NIR-I) imaging to NIR-II imaging, and from one-mode to dual-mode of chemo-fluoro-luminescence sensing β-gal activity.

This review highlights the molecular design strategy of β-galactosidase-activatable probes from turn-on mode to ratiometric mode, from ACQ to AIE-active probes, from NIR-I to NIR-II imaging and dual-mode of chemo-fluoro-luminescence imaging.     

Determination of Polycyclic Aromatic Hydrocarbons in Traditional Chinese Medicine Raw Material,Extracts, and Health Food Products

The four polycyclic aromatic hydrocarbon markers (PAH4) of benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), and benzo[a]pyrene (BaP) are indicators showing polycyclic aromatic hydrocarbon (PAH) contamination levels in Chinese medicine raw materials (CMRMs), extracts and health food products; Samples of herbal medicine, herbal extracts, and food supplements were extracted with n-hexane, then cleaned up sequentially on Florisil and EUPAH solid-phase extraction (SPE) columns. A gas chromatography–mass spectrometry method for the determination of four polycyclic aromatic hydrocarbon markers in Chinese medicine raw material, extracts, and health food products was established; In spiked-recovery experiments, the average recovery was about 78.6–107.6% with a precision of 2.3–10.5%. The limit of quantification (LOQ) and limit of detection (LOD) of the PAH4 markers in this method were 2.0 μg/kg and 0.7 μg/kg, respectively. When the developed method was utilized to determine PAH4 contents in 12 locally available health food products, 3 samples contained over 10.0 μg/kg BaP, and 5 samples contained over 50.0 μg/kg PAH4. The European Union (EU) limits for BaP and PAH4 are 10 and 50.0 μg/kg, respectively; therefore, more attention must be drawn to the exposure risk of BaP and PAH4 in CMRMs, their extracts, and health food products. According to the risk assessment based on the Margin of Exposure (MOE) method, it is recognized that the products mentioned in this study pose a low risk.     

Harnessing α-l-fucosidase for in vivo cellular senescence imaging

Precise detection of cellular senescence may allow its role in biological systems to be evaluated more effectively, while supporting studies of therapeutic candidates designed to evade its detrimental effect on physical function. We report here studies of α-l-fucosidase (α-fuc) as a biomarker for cellular senescence and the development of an α-fuc-responsive aggregation induced emission (AIE) probe, termed QM-NHαfuc designed to complement more conventional probes based on β-galactosidase (β-gal). Using QM-NHαfuc, the onset of replicative-, reactive oxygen species (ROS)-, ultraviolet A (UVA)-, and drug-induced senescence could be probed effectively. QM-NHαfuc also proved capable of identifying senescent cells lacking β-gal expression. The non-invasive real-time senescence tracking provided by QM-NHαfuc was validated in an in vivo senescence model. The results presented in this study lead us to suggest that the QM-NHαfuc could emerge as a useful tool for investigating senescence processes in biological systems.

Evidence of close association of α-fuc with senescence induction highlights the potential of α-fuc as a novel biomarker for cellular senescence. Here, an α-fuc-responsive AIE probe (QM-NHαfuc) allows for the identification of senescent cell in vivo.     

Design and Functionalization of a µPAD for the Enzymatic Determination of Nitrate in Urine

In this work, the design of a microfluidic paper-based analytical device (μPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination μPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed μPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14–1.0 mM. The designed μPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at −20 °C. The accuracy of the nitrate μPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.     

A combination therapy strategy for treating antibiotic resistant biofilm infection using a guanidinium derivative and nanoparticulate Ag(0) derived hybrid gel conjugate

Bacteria organized in biofilms show significant tolerance to conventional antibiotics compared to their planktonic counterparts and form the basis for chronic infections. Biofilms are composites of different types of extracellular polymeric substances that help in resisting several host-defense measures, including phagocytosis. These are increasingly being recognized as a passive virulence factor that enables many infectious diseases to proliferate and an essential contributing facet to anti-microbial resistance. Thus, inhibition and dispersion of biofilms are linked to addressing the issues associated with therapeutic challenges imposed by biofilms. This report is to address this complex issue using a self-assembled guanidinium–Ag(0) nanoparticle (AD-L@Ag(0)) hybrid gel composite for executing a combination therapy strategy for six difficult to treat biofilm-forming and multidrug-resistant bacteria. Improved efficacy was achieved primarily through effective biofilm inhibition and dispersion by the cationic guanidinium ion derivative, while Ag(0) contributes to the subsequent bactericidal activity on planktonic bacteria. Minimum Inhibitory Concentration (MIC) of the AD-L@Ag(0) formulation was tested against Acinetobacter baumannii (25 μg mL⁻¹), Pseudomonas aeruginosa (0.78 μg mL⁻¹), Staphylococcus aureus (0.19 μg mL⁻¹), Klebsiella pneumoniae (0.78 μg mL⁻¹), Escherichia coli (clinical isolate (6.25 μg mL⁻¹)), Klebsiella pneumoniae (clinical isolate (50 μg mL⁻¹)), Shigella flexneri (clinical isolate (0.39 μg mL⁻¹)) and Streptococcus pneumoniae (6.25 μg mL⁻¹). Minimum bactericidal concentration, and MBIC₅₀ and MBIC₉₀ (Minimum Biofilm Inhibitory Concentration at 50% and 90% reduction, respectively) were evaluated for these pathogens. All these results confirmed the efficacy of the formulation AD-L@Ag(0). Minimum Biofilm Eradication Concentration (MBEC) for the respective pathogens was examined by following the exopolysaccharide quantification method to establish its potency in inhibition of biofilm formation, as well as eradication of mature biofilms. These effects were attributed to the bactericidal effect of AD-L@Ag(0) on biofilm mass-associated bacteria. The observed efficacy of this non-cytotoxic therapeutic combination (AD-L@Ag(0)) was found to be better than that reported in the existing literature for treating extremely drug-resistant bacterial strains, as well as for reducing the bacterial infection load at a surgical site in a small animal BALB/c model. Thus, AD-L@Ag(0) could be a promising candidate for anti-microbial coatings on surgical instruments, wound dressing, tissue engineering, and medical implants.

Dispersion of biofilms that protect bacteria and its subsequent killing in the planktonic state are effectively achieved by a guanidinium–Ag(0) nanocomposite.     

High-efficiency dynamic sensing of biothiols in cancer cells with a fluorescent β-cyclodextrin supramolecular assembly

A unique fluorescent supramolecular assembly was constructed using coumarin-modified β-cyclodextrin as a reversible ratiometric probe and an adamantane-modified cyclic arginine–glycine–aspartate peptide as a cancer-targeting agent via host–guest inclusion complexation. Importantly, the coumarin-modified β-cyclodextrin not only showed higher sensitivity than the parent coumarin derivatives owing to the presence of numerous hydroxyl groups on the cyclodextrin but also provided a hydrophobic cavity for encapsulation of a cancer-targeting agent. The assembly showed a reversible and fast response to biothiols with a micromolar dissociation constant, as well as outstanding cancer cell permeability, which can be used for high-efficiency real-time monitoring of biothiols in cancer cells. This supramolecular assembly strategy endows the fluorescent probe with superior performance for dynamic sensing of biothiols.

A unique fluorescent supramolecular assembly was constructed from coumarin-modified β-cyclodextrin and an adamantane-modified cyclic arginine–glycine–aspartate peptide for high-efficiency real-time monitoring of biothiols in cancer cells.     

Ratiometric two-photon excited photoluminescence of quantum dots triggered by near-infrared-light for real-time detection of nitric oxide release in situ

Probe-donor integrated nanocomposites were developed from conjugating silica-coated Mn²⁺:ZnS quantum dots (QDs) with MoS₂ QDs and photosensitive nitric oxide (NO) donors (Fe₄S₃(NO)₇⁻, RBS). Under excitation with near-infrared (NIR) light at 808 nm, the Mn²⁺:ZnS@SiO₂/MoS₂-RBS nanocomposites showed the dual-emissive two-photon excited photoluminescence (TPEPL) that induced RBS photolysis to release NO in situ. NO caused TPEPL quenching of Mn²⁺:ZnS QDs, but it produced almost no impact on the TPEPL of MoS₂ QDs. Hence, the nanocomposites were developed as a novel QDs-based ratiometric TPEPL probe for real-time detection of NO release in situ. The ratiometric TPEPL intensity is nearly linear (R² = 0.9901) with NO concentration in the range of 0.01∼0.8 μM, which corresponds to the range of NO release time (0∼15 min). The detection limit was calculated to be approximately 4 nM of NO. Experimental results confirmed that this novel ratiometric TPEPL probe possessed high selectivity and sensitivity for the detection of NO against potential competitors, and especially showed high detection performance for NIR-light triggered NO release in tumor intracellular microenvironments. These results would promote the development of versatile probe-donor integrated systems, also providing a facile and efficient strategy to real-time detect the highly controllable drug release in situ, especially in physiological microenvironments.     

An “OFF–ON–OFF” fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an ‘OFF–ON–OFF’ fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag–probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting ‘OFF–ON–OFF’ probe to be used to fluorescently image the expression and degradation of short-lived proteins.

An “OFF–ON–OFF” fluorescence probe for real-time imaging of the expression (fluorescence ‘OFF’) and degradation (fluorescence ‘ON’) of short lived PYP-tag proteins in cellular systems.     

A Simple HPLC/DAD Method Validation for the Quantification of Malondialdehyde in Rodent’s Brain

In the present study, a HPLC/DAD method was set up to allow for the determination and quantification of malondialdehyde (MDA) in the brain of rodents (rats). Chromatographic separation was achieved on Supelcosil LC-18 (3 μm) SUPELCO Column 3.3 cm × 4.6 mm and Supelco Column Saver 0.5 μm filter by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 6) (B). Isocratic elution was 14% for (A) and 86% for (B). The injection volume (loop mode) was 100 μL with an analysis time of 1.5 min. Flow rate was set at 1 mL/min. The eluted compound was detected at 532 nm by a DAD detector by keeping the column oven at room temperature. The results indicated that the method has good linearity in the range of 0.2–20 μg/g. Both intra- and inter-day precision, expressed as RSD, were ≤15% and the accuracies ranged between ±15%. The lower limit of quantification (LLOQ), stability, and robustness were evaluated and satisfied the validation criteria. The method was successfully applied in a study of chronic toxicology following different treatment regimens with haloperidol and metformin.     

Dual electrochemical microsensor for simultaneous measurements of nitric oxide and oxygen: Fabrication and characterization

A planar-type amperometric dual microsensor was developed for the simultaneous measurement of the nitric oxide (NO) and oxygen (O₂) concentrations. The sensor (overall diameter = 500 μm) consisted of a dual working electrode (WE) containing two platinized platinum microdisks (25 μm diameter, WE1, WE2, distance between two disks > 330 μm) and a Ag/AgCl wire reference electrode covered with an expanded poly(tetrafluoroethylene) gas-permeable membrane. The differentiation and concurrent measurements of NO and O₂ were obtained successfully using two sensing WEs with different applied potentials (+0.75 V for WE1 and −0.4 V for WE2). Cross-talk between WE1 and WE2 was eliminated with an optimized internal solution composition. Linear dynamic range, selectivity, sensitivity, detection limit (<5 nM for NO; <500 nM for O₂), and stability (>50 h) were evaluated.     

Azodioxy compounds as precursors for C-radicals and their application in thermal styrene difunctionalization

An atom-economic thermal α,β-difunctionalization of various styrenes with readily prepared azodioxy compounds is reported. Mechanistic studies reveal that the starting azodioxy compounds can thermally be cleaved to the corresponding C-nitroso compounds, which under these thermal conditions further homolyze to generate reactive C-radicals along with the persistent NO radical. In the presence of a styrene, C-radical addition with subsequent nitrosylation followed by tautomerization is occurring, resulting in an overall styrene β-alkylation-α-oximation reaction.

An atom-economic thermal α,β-difunctionalization of various styrenes with readily prepared azodioxy compounds is reported.     

In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva''s caudal fin regeneration.     

Locally superengineered cascade recognition–quantification zones in nanochannels for sensitive enantiomer identification

As an intriguing and intrinsic feature of life, chirality is highly associated with many significant biological processes. Simultaneous recognition and quantification of enantiomers remains a major challenge. Here, a sensitive enantiomer identification device is developed on TiO₂ nanochannels via the design of cascade recognition–quantification zones along the nanochannels. In this system, β-cyclodextrin (β-CD) is self-assembled on one side of the nanochannels for the selective recognition of enantiomers; CuMOFs are designed as the target-responsive partners on the other side of the nanochannels for the quantification of enantiomers that pass through the nanochannels. As a proof-of-principle of the cascade design, arginine (Arg) enantiomers are tested as the identification targets. The l-Arg molecules selectively bind in the recognition zone; d-Arg molecules pass through the recognition zone and then interact with the quantification zone via a specialized reduction reaction. As verified by nanofluidic simulations, because of the confinement effect of nanoscale channels combined with the condensation effect of porous structure, the in situ reaction in the quantification zone contributes to an unprecedented variation in transmembrane K⁺ flux, leading to an improved identification signal. This novel cascade-zone nanochannel membrane provides a smart strategy to design multifunctional nanofluidic devices.

A design of the cascade recognition–quantification zone is developed along TiO₂ nanochannels. The asymmetric nanochannels exhibit a predominant sensitivity and selectivity for enantiomer discrimination.     

Unravelling the last milliseconds of an individual graphene nanoplatelet before impact with a Pt surface by bipolar electrochemistry

Contactless interactions of micro/nano-particles near electrochemically or chemically active interfaces are ubiquitous in chemistry and biochemistry. Forces arising from a convective field, an electric field or chemical gradients act on different scales ranging from few microns down to few nanometers making their study difficult. Here, we correlated optical microscopy and electrochemical measurements to track at the millisecond timescale the dynamics of individual two-dimensional particles, graphene nanoplatelets (GNPs), when approaching an electrified Pt micro-interface. Our original approach takes advantage of the bipolar feedback current recorded when a conducting particle approaches an electrified surface without electrical contact and numerical simulations to access the velocity of individual GNPs. We evidenced a strong deceleration of GNPs from few tens of μm s⁻¹ down to few μm s⁻¹ within the last μm above the surface. This observation reveals the existence of strongly non-uniform forces between tens of and a thousand nanometers from the surface.

The velocity of single GNP is monitored by contactless bipolar electrochemical feedback over the last hundreds of nm before collision on an electrode, and the variations shed light on the balance of forces acting on these objects near an interface.     

Excitation ratiometric chloride sensing in a standalone yellow fluorescent protein is powered by the interplay between proton transfer and conformational reorganization

Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl⁻). To date, such sensors have been limited to the Aequorea victoria green fluorescent protein (avGFP) family, and fusions with other FPs have unlocked ratiometric imaging applications. Recently, we identified the yellow fluorescent protein from jellyfish Phialidium sp. (phiYFP) as a fluorescent turn-on, self-ratiometric Cl⁻ sensor. To elucidate its working mechanism as a rare example of a single FP with this capability, we tracked the excited-state dynamics of phiYFP using femtosecond transient absorption (fs-TA) spectroscopy and target analysis. The photoexcited neutral chromophore undergoes bifurcated pathways with the twisting-motion-induced nonradiative decay and barrierless excited-state proton transfer. The latter pathway yields a weakly fluorescent anionic intermediate , followed by the formation of a red-shifted fluorescent state that enables the ratiometric response on the tens of picoseconds timescale. The redshift results from the optimized π–π stacking between chromophore Y66 and nearby Y203, an ultrafast molecular event. The anion binding leads to an increase of the chromophore pK_a and ESPT population, and the hindrance of conversion. The interplay between these two effects determines the turn-on fluorescence response to halides such as Cl⁻ but turn-off response to other anions such as nitrate as governed by different binding affinities. These deep mechanistic insights lay the foundation for guiding the targeted engineering of phiYFP and its derivatives for ratiometric imaging of cellular chloride with high selectivity.

We discovered an interplay between proton transfer and conformational reorganization that powers a standalone fluorescent-protein-based excitation-ratiometric biosensor for chloride imaging.     

A long-lived charge-separated state of spiro compact electron donor–acceptor dyads based on rhodamine and naphthalenediimide chromophores

Spiro rhodamine (Rho)-naphthalenediimide (NDI) electron donor–acceptor orthogonal dyads were prepared to generate a long-lived charge separation (CS) state based on the electron spin control approach, i.e. to form the ³CS state, not the ¹CS state, to prolong the CS state lifetime by the electron spin forbidden feature of the charge recombination process of ³CS → S₀. The electron donor Rho (lactam form) is attached via three σ bonds, including two C–C and one N–N bonds (Rho-NDI), or an intervening phenylene, to the electron acceptor NDI (Rho-Ph-NDI and Rho-PhMe-NDI). Transient absorption (TA) spectra show that fast intersystem crossing (ISC) (<120 fs) occurred to generate an upper triplet state localized on the NDI moiety (³NDI*), and then to form the CS state. For Rho-NDI in both non-polar and polar solvents, a long-lived ³CS state (lifetime τ = 0.13 μs) and charge separation quantum yield (Φ_CS) up to 25% were observed, whereas for Rho-Ph-NDI (τ_T = 1.1 μs) and Rho-PhMe-NDI (τ_T = 2.0 μs), a low-lying ³NDI* state was formed by charge recombination (CR) in n-hexane (HEX). In toluene (TOL), however, CS states were observed for Rho-Ph-NDI (0.37 μs) and Rho-PhMe-NDI (0.63 μs). With electron paramagnetic resonance (EPR) spectra, weak electronic coupling between the Rho and NDI moieties for Rho-NDI was proved. Time-resolved EPR (TREPR) spectra detected two transient species including NDI-localized triplets (formed via SOC-ISC) and a ³CS state. The CS state of Rho-NDI features the largest dipolar interaction (|D| = 184 MHz) compared to Rho-Ph-NDI (|D| = 39 MHz) and Rho-PhMe-NDI (|D| = 41 MHz) due to the smallest distance between Rho and NDI moieties. For Rho-NDI, the time-dependent e,a → a,e phase change of the CS state TREPR spectrum indicates that the long-lived CS state is based on the electron spin control effect.

Spiro compact rhodamine-naphthalenediimide electron donor–acceptor dyads show a long-lived charge separated state (lifetime: 0.72 μs) based on the electron spin control effect were reported.     

Ultrasensitive detection of β-lactamase-associated drug-resistant bacteria using a novel mass-tagged probe-mediated cascaded signal amplification strategy

The emergence and spread of drug-resistant bacteria (DRB) is a global health threat. Early and accurate detection of DRB is a critical step in the treatment of DRB infection. However, traditional assays for DRB detection are time-consuming and have inferior analytical sensitivity and quantification capability. Herein, a mass-tagged probe (MP-CMSA)-mediated enzyme- and light-assisted cascaded signal amplification strategy was developed for the ultrasensitive detection of β-lactamase (BLA), an enzyme closely associated with most DRB. Each MP-CMSA probe contained multiple poly(amidoamine) (PAMAM) dendrimer molecules immobilized on a streptavidin agarose bead via a BLA-cleavable linker, and each dendrimer was modified with multiple mass tags via a photo-cleavable linker. In BLA detection, BLA could cleave the BLA-cleavable linker, leading to dendrimers shedding from the MP-CMSA probe to achieve enzyme-assisted signal amplification. Then, each dendrimer can further release mass tags under UV light to achieve light-assisted signal amplification. After this cascaded signal amplification, the released mass tags were ultimately quantified by mass spectrometry. Consequently, the sensitivity of BLA detection can be significantly enhanced by four orders of magnitude with a detection limit of 50.0 fM. Finally, this approach was applied to the blood samples from patients with DRB. This platform provides a potential strategy for the sensitive, rapid and quantitative detection of DRB infection.

Development of a mass-tagged probe-mediated enzyme- and light-assisted cascaded signal amplification strategy for the ultrasensitive detection of β-lactamase.     

Fabrication of a Ratiometric Fluorescence Sensor Based on Carbon Dots as Both Luminophores and Nanozymes for the Sensitive Detection of Hydrogen Peroxide

The construction of novel fluorescent nanozymes is highly desirable for providing new strategies for nanozyme-based sensing systems. Herein, a novel ratiometric fluorescence sensing platform was constructed based on carbon dots (CDs) as both luminophores and nanozymes, which could realize the sensitive detection of hydrogen peroxide (H₂O₂). CDs with peroxidase-mimicking activity were prepared with a one-step hydrothermal method using L-histidine as an inexpensive precursor. CDs had bright blue fluorescence. Due to the pseudo-peroxidase activity, CDs catalyzed the oxidation of o-phenylenediamine (OPD) with H₂O₂ to generate 2,3-diaminophenolazine (DAP). The fluorescence resonance energy transfer (FRET) between CDs and DAP resulted in a decrease in the fluorescence of CDs and an increase in the fluorescence of DAP, leading to a ratiometric fluorescence system. The free radical trapping experiment was used to investigate the reactive oxygen radicals (ROS) in the catalytic process of CD nanozymes. The enzymatic parameters of CD nanozymes, including the Michaelis constant (K_m) and the maximum initial reaction velocities (V_max), were investigated. A good affinity for both OPD and H₂O₂ substrates was proven. Based on the FRET between CDs and OPD, a ratiometric fluorescence analysis of H₂O₂ was achieved and results ranged from 1 to 20 μM and 20 to 200 μM with a low limit of detection (LOD, 0.42 μM). The detection of H₂O₂ in milk was also achieved.