Ultrasmall CuMn-His Nanozymes with Multienzyme Activity at Neutral pH: Construction of a Colorimetric Sensing Array for Biothiol Detection and Disease Identification.

Biothiol assays offer vital insights into health assessment and facilitate the early detection of potential health issues, thereby enabling timely and effective interventions. In this study, we developed ultrasmall CuMn-Histidine (His) nanozymes with multiple enzymatic activities. CuMn-His enhanced peroxidase (POD)-like activity at neutral pH was achieved through hydrogen bonding and electrostatic effects. In addition, CuMn-His possesses laccase (LAC)-like and superoxide dismutase (SOD)-like activities at neutral pH. Based on three different enzyme mimetic activities of CuMn-His at neutral pH, the colorimetric sensing array without changing the buffer solution was successfully constructed. The array was successfully used for the identification of three biothiols, glutathione (GSH), cysteine (Cys), and homocysteine (Hcy). Subsequently, excellent application results were shown in complex serum and cellular level analyses. This study provides an innovative strategy for the development of ultrasmall bimetallic nanozymes with multiple enzymatic activities and the construction of colorimetric sensing arrays.

Peroxidase-like activity and mechanism of gold nanoparticle-modified Ti3C2 MXenes for the construction of H2O2 and ampicillin colorimetric sensors.

Transition metal carbides modified by Au nanoparticles (Au/Ti3C2) were synthesized and developed as a colorimetric sensor for the determination of H2O2 and ampicillin. The surface electrical properties of Ti3C2 were changed, and Au nanoparticles (AuNPs) and gold growth solution were synthesized simultaneously. Au/Ti3C2 was obtained by seed growth method with AuNPs modified on the surface of transition metal carbides, nitrides or carbon-nitrides (Ti3C2 MXenes). The synthesized AuNPs and Ti3C2 had no peroxidase-like activity, but Au/Ti3C2 had. The peroxidase catalytic mechanism was due to electron transfer. The peroxidase activity of Au/Ti3C2 can be utilized for the determination of H2O2. The linear range of Au/Ti3C2 for H2O2 was 1-60 µM, and the detection limit was 0.12 µM (S/N = 3). A colometric sensor for ampicillin detection based on Au/Ti3C2 was further constructed since S in ampicillin formed an Au-S bond with Au/Ti3C2, leading to the weakening of its peroxidase-like property. The change of peroxidase-like property attenuated oxidation of TMB, and the ampicillin content was inversely proportional to the concentration of oxidized TMB, and the blue color of solution faded, which enabled the determination of ampicillin. The linear range for ampicillin was 0.005-0.5 µg mL- 1, and the detection limit was 1.1 ng mL- 1 (S/N = 3). The sensor was applied to the detection of ampicillin in milk and human serum.

Ultrasmall Cu2O@His Nanozymes with RONS Scavenging Capability for Anti-inflammatory Therapy.

High concentrations of reactive oxygen and nitrogen species (RONS) are key characteristics of inflammatory sites. Scavenging RONS at the site of inflammation is an effective therapeutic strategy. This study introduces ultrasmall Cu2O@His nanoparticles with RONS-scavenging ability for the treatment of inflammatory bowel disease (IBD) in mice. The strong coordination between the nitrogen atom in histidine (His) and copper enhances the dispersion and stability of Cu2O@His. Due to their small size and large surface area, Cu2O@His exhibits outstanding RONS-clearing ability. Importantly, Cu2O@His can target mitochondrial sites and repair damaged mitochondria. With excellent dispersion and scavenging RONS ability, Cu2O@His demonstrates good efficacy in treating mouse IBD. This work provides a new paradigm for developing nanozymes with an ultrasmall size and multiple scavenging RONS abilities.

Self-template sacrifice and in situ oxidation of a constructed hollow MnO2 nanozymes for smartphone-assisted colorimetric detection of liver function biomarkers.

Liver function tests play a vital role in accurately diagnosing liver diseases, monitoring treatment outcomes, and assessing liver damage severity. Here, we introduce a novel approach to develop a smartphone-assisted portable colorimetric sensor for rapid detection of three liver function biomarkers: aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). This sensor is based on the inherent enzyme-like activities of hollow MnO2 (H-MnO2). The H-MnO2 is synthesized via a self-template sacrifice and in situ oxidation strategy, utilizing a manganese-based Prussian blue analogue (Mn-PBA) as a sacrificial template. The resulting H-MnO2 exhibits a polycrystalline structure with a large specific surface area. By encapsulating the H-MnO2 in sodium alginate, we construct a portable sensing platform facilitating specific and rapid colorimetric detection of the three liver function biomarkers with the assistance of a smartphone. The developed sensor demonstrates outstanding sensitivity and stability, achieving detection limits of 4.9 U L-1, 3.6 U L-1, and 0.99 U L-1 for AST, ALT, and ALP, respectively. Importantly, this work introduces an innovative in situ oxidation method for fabricating hollow nanozymes, offering a cost-effective and convenient assay for liver function biomarkers detection.

Peroxidase-mimicking poly-L-lysine/alginate microspheres with PtS2 nanoparticles for image-based colorimetric assays.

Morphologically controllable ALG@ε-PL water-in-water microspheres were successfully prepared using a two-step method through precise control of the two-phase flow rate. Through further interfacial coagulation, the ALG@ε-PL microspheres possess a dense surface structure and good permeability. The sensor based on PtS2@ALG@ε-PL microspheres was constructed by encapsulating PtS2 nanosheets with peroxidase-like properties in ALG@ε-PL water-in-water microspheres. PtS2 nanosheets catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce blue oxTMB. The strong reducing property of the model analyte dopamine (DA) can reduce oxTMB, thus causing the blue color to fade and successfully achieving colorimetric detection of DA. The linear range of the assay is 2.0-200 µM, and the detection limit is 0.22 µM. The recoveries of DA in serum samples were determined by the spik method, and the results were reproducible.

Fluorescent sensor based on PtS2-PEG nanosheets with peroxidase-like activity for intracellular hydrogen peroxide detection and imaging.

Hydrogen peroxide (H2O2) is produced in living organisms and is involved in a variety of redox-regulated processes. Therefore, the detection of H2O2 is important for tracing the molecular mechanisms of some biological events. Here, we demonstrated for the first time the peroxidase activity of PtS2-PEG NSs under the physiological conditions. PtS2 NSs were synthesized by mechanical exfoliation followed by functionalization with polyethylene glycol amines (PEG-NH2) to improve their biocompatibility and physiological stability. Fluorescence was generated by catalyzing the oxidation of o-phenylenediamine (OPD) by H2O2 in the presence of the PtS2 NSs. The proposed sensor had a limit of detection (LOD) of 248 nM and a detection range of 0.5-50 µM in the solution state, which was better than or comparable to previous reports in the literature. The developed sensor was further applied for the detection of H2O2 released from cells as well as for imaging studies. The results show that the sensor is promising for future applications in clinical analysis and pathophysiology.

Injectable and Self-Healing Hydrogel Based on Chitosan-Tannic Acid and Oxidized Hyaluronic Acid for Wound Healing.

Self-healing performance plays an important role in the in situ microinvasive injection of hydrogels, which can reduce sudden drug release and prolong the service life of hydrogels. In this paper, a multifunctional injectable and self-healing hydrogel for wound healing was developed. Chitosan (CS) was modified with TA to achieve potential adhesion, anti-inflammatory properties, and slower degradation rate. The hydrogel was formed by Schiff base reaction based on amino groups in CS and aldehyde groups in oxidized hyaluronic acid (OHA). The gel formation process was quick and convenient in mild conditions without extra initiators. Due to the dynamically reversible covalent bonds, the hydrogel could self-heal within 2 min after injection. It also had good biocompatibility and hemostatic performance. With the addition of TA, the hydrogel acquired anti-inflammatory properties and promoted cell growth, effectively accelerating the wound-healing process in vivo. The CS-TA/OHA hydrogel is expected to be used for skin repair.

Molecular design of peptide amphiphiles for controlled self-assembly and drug release.

Peptide amphiphile-based supramolecular hydrogels hold great promise in drug delivery applications. To cater for a specific drug dose in a demanding biomedical scenario, sophisticated design of peptide amphiphile (PA) molecules is required to tune their self-assembling behaviours as well as drug releasing profiles. Herein, we designed a series of PAs with various capping groups and C-terminal amino acids to systematically optimize their self-assembling capabilities for controlled drug release. First, we evaluated the influence of N-terminal capping groups to find that the 2-naphthylacetyl moiety (Nap) greatly assisted hydrogelation of PAs. Next, self-assembling behaviours of Nap-capped PAs were compared among three candidates that bore varying hydrophilic moieties at the C-terminus (Nap-C12-VVAAG, Nap-C12-VVAAD and Nap-C12-VVAADD, denoted as 1-G, 1-D, and 1-DD). It was found that 1-G and 1-D co-assembled with doxorubicin (DOX) and calcium ions (Ca2+) at a higher efficiency than 1-DD, for 1-G/Ca2+/DOX and 1-D/Ca2+/DOX hydrogels displayed a dense nanofibrillar network, with lower minimal gelation concentrations and greater storage modulus values. Interestingly, these PA/Ca2+/DOX hydrogels exhibited tunable release rates of DOX in vitro, with fast release of DOX found in 1-DD/Ca2+/DOX and slow release in 1-G/Ca2+/DOX and 1-D/Ca2+/DOX. Further cell experiments demonstrated that 1-G/Ca2+/DOX and 1-D/Ca2+/DOX exhibited higher inhibitory efficacy against HeLa cells, as compared to DOX solution and 1-DD/Ca2+/DOX. Finally, PA/Ca2+/DOX hydrogels displayed a longer retention time of DOX than aqueous DOX solution in animal experiments, and sustained release of DOX from hydrogels was also evidenced by slow and persisting accumulation of DOX in the major organs of hydrogel-treated mice.

PtS2 nanosheets as a peroxidase-mimicking nanozyme for colorimetric determination of hydrogen peroxide and glucose.

Using an ultrasonication-assisted liquid exfoliation method, we have synthesized PtS2 nanosheets with good reproducibility. Herein, intrinsic peroxidase-like activity was for the first time demonstrated for PtS2 nanosheets, which can catalyze H2O2 oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate a colored solution. The catalytic mechanism of PtS2 nanosheets was investigated, which indicated that acceleration of the electron transfer between TMB and H2O2 was the main reason for the peroxidase-like activity of PtS2 nanosheets. Based on these observations, we exploited PtS2 nanosheets integrated into dopamine-functionalized hyaluronic acid (HA-DA) hydrogel microspheres by droplet microfluidics to construct PtS2 nanosheet- and PtS2@HA-DA microsphere-based sensors for highly sensitive determination of H2O2. When coupled with glucose oxidase, we further developed two glucose sensors based on the above two methods. Among them, the linearity of the PtS2 nanosheet-based spectrophotometry was in the range of 0.5 to 150 µM and the limit of detection as low as 0.20 µM. The linearity of the microsphere-based colorimetry was in the range 200 to 12,000 µM with a detection limit of 29.95 µM. Both of the glucose sensors can be applied to the determination of glucose in human serum with reliable results and reproducibility.

Dopamine-functionalized hyaluronic acid microspheres for effective capture of CD44-overexpressing circulating tumor cells.

As one of the biomarkers of liquid biopsy, circulating tumor cells (CTCs) provides important clinical information for cancer diagnosis. However, accurate separation and identification of CTCs remains a great deal of challenge. In present work, we developed novel dopamine-functionalized hyaluronic acid microspheres (HA-DA microspheres) to capture CD44-overexpressing CTCs. The dopamine was grafted onto the hyaluronic acid chain, which was polymerized and cross-linked by oxidation of the catechol groups. Afterwards, a facile microfluidic chip was designed and developed to fabricate the HA-DA microspheres with a diameter of about 45 µm. Our results showed that the CD44+ cells (i.e., HeLa, HepG2, A549, MCF-7 and DU-145 cells) could be selectively captured. Then a double-layer microfluidic filter (DLMF) was fabricated for dynamic isolation and detection of CTCs in blood samples. Many slit openings with 15 µm in height were designed to allow white blood cells to clear away, while the microspheres with CTCs were intercepted in the DLMF, which achieved effective separation of CTCs from blood cells. The approach exhibited high capture efficiency even at the cell density as low as 10 cells/mL. We believe the DLMF integrated with HA-DA microspheres could be a promising approach for isolation and detection of CD44-overexpressing CTCs, which is useful for prognosis and early metastasis of cancer patients.

PEG-PtS2 nanosheet-based fluorescence biosensor for label-free human papillomavirus genotyping.

A simple and efficient ultrasonication-assisted liquid exfoliation method is proposed to produce PtS2 nanosheets on a large scale and improve their dispersion in aqueous solution by surface polyethylene glycol modification. The interaction of polyethylene glycol-modified PtS2 (PEG-PtS2) nanosheets with fluorescent labeled DNA and the fluorescence quenching mechanism using FAM-labeled hpv16e6 gene fragment as a probe was investigated. The excitation and emission wavelengths were 468 and 517 nm, respectively. The fluorescence quenching mechanism of PEG-PtS2 nanosheets for double-stranded DNA (dsDNA) might stem from the static quenching effect. Based on the difference in fluorescence quenching capability of PEG-PtS2 nanosheets in fluorescent probe tagged single-stranded DNA (ssDNA) and dsDNA, a mix-and-detect method was proposed for determination of DNA. Without the need for probe immobilization and tedious washing steps, the genotyping of human papillomavirus (HPV) was easily achieved. The limit of detection was calculated to 0.44 nM, showing a good linear range within 0.05-10 nM. We believe this biosensor provides opportunities to develop a simple and low-cost strategy for molecular diagnostics. Graphical abstract.

Instant Self-Assembly Peptide Hydrogel Encapsulation with Fibrous Alginate by Microfluidics for Infected Wound Healing.

Infected wounds caused by persistent inflammation exhibit poor vascularization and cellular infiltration. In order to rapidly control the inflammatory effect and accelerate wound healing, it is necessary to develop a novel drug vehicle addressing the need for infected wounds. Herein, we developed a novel dual-drug delivery system with micrometer-scale alginate fibers encapsulated in instant self-assembly peptide hydrogel. Short peptides with the sequence of Nap-Gly-Phe-Phe-Lys-His (Nap-GFFKH) could self-assemble outside the microfluidic-based alginate microfibers in weak acidic solution (pH ≈ 6.0) within 5 s. The gelation condition is close to the pH environment of the human skin. We further constructed recombinant bovine basic fibroblast growth factor (FGF-2) in fibrous alginate, which was encapsulated in antibiotic-loaded peptide hydrogel. The dual-drug delivery system exhibited good mechanical property and sustained release profiles, where antibiotic could be rapidly released from the peptide hydrogel, while the growth factor could be gradually released within 7 days. Both in vitro antibacterial experiments and in vivo animal experiments confirmed that such a dual-drug delivery system has good antibacterial activity and enhances wound healing property. We suggested that the dual-drug delivery system could be potentially applied for controlled drug release in infected wound healing, drug combination for melanoma therapy, and tissue engineering.

A simply triggered peptide-based hydrogel as an injectable nanocarrier of tanshinone IIA and tanshinones.

An easily self-assembled and gelated octa-peptide FHFDFHFD was chosen as a novel drug delivery system (DDS) for both tanshinone IIA and total tanshinone extract. The DDS showed increased loading capacity, sustained drug release and better anticancer capability. Our research proved that a hydrogel DDS of other traditional Chinese medicines is possible.

Monitoring the intracellular calcium response to a dynamic hypertonic environment.

The profiling of physiological response of cells to external stimuli at the single cell level is of importance. Traditional approaches to study cell responses are often limited by ensemble measurement, which is challenging to reveal the complex single cell behaviors under a dynamic environment. Here we report the development of a simple microfluidic device to investigate intracellular calcium response to dynamic hypertonic conditions at the single cell level in real-time. Interestingly, a dramatic elevation in the intracellular calcium signaling is found in both suspension cells (human leukemic cell line, HL-60) and adherent cells (lung cancer cell line, A549), which is ascribed to the exposure of cells to the hydrodynamic stress. We also demonstrate that the calcium response exhibits distinct single cell heterogeneity as well as cell-type-dependent responses to the same stimuli. Our study opens up a new tool for tracking cellular activity at the single cell level in real time for high throughput drug screening.

Rapid determination of dopamine in human plasma using a gold nanoparticle-based dual-mode sensing system.

Dopamine plays a very important role in biological systems and has a direct relationship with the ability of learning and cognition, human desires, feelings and mental state, as well as motor functions. Traditional methods for the detection of dopamine are complicated and time-consuming, therefore it is necessary to explore rapid and accurate detection of dopamine with high sensitivity and specificity. Herein we report a dual-mode system of colorimetric and fluorometric analyses based on gold nanoparticles (AuNPs) and aptamers specifically targeting dopamine. Aptamers modified with the fluorophore were used as dopamine specific recognition probe and the sensing mechanism is based on the color change of AuNPs and the fluorescence recovery of fluorophore conjugated on the aptamers in the presence of dopamine. The addition of aptamers into AuNPs colloid solution would prevent the AuNPs from aggregation in the high-salt solution. The close distance between AuNPs and fluorophore conjugated on the aptamers would lead to the quenching of fluorescence signal. In the presence of dopamine, the conformation of the aptamers and the inter-particle distance would be changed, leading to the aggregation of AuNPs, which subsequently results in color change from red to blue and fluorescence signal recovery. The dual-mode sensing system demonstrated high specificity towards dopamine with the detection limit as low as 78.7 nM. The sensing system reflects on its simplicity as no surface functionalization is required for the nanoparticles, leading to less laborious and more cost-effective synthesis. The reaction time is only 6 min, demonstrating a simple approach for rapid analysis of dopamine. More importantly, the sensing system allows the detection of dopamine in both aqueous solution and complicated biological sample with sensitive response, illustrating the feasibility and reliability for the potential applications in clinical and biomedical analysis in the future.

Overcoming multidrug resistance by a combination of chemotherapy and photothermal therapy mediated by carbon nanohorns.

Multidrug resistance (MDR) is a major obstacle to cancer chemotherapy due to the overexpression of P-glycoprotein (P-gp). Herein, etoposide (ETO) was loaded onto oxidized carbon nanohorns (oxCNHs), which were modified by polyethylene glycol (PEG) and further functionalized with the targeting ligand P-gp monoclonal antibody (PA) in an attempt to overcome MDR. The obtained drug delivery system (ETO@oxCNHs/PEG-PA) showed high drug loading efficiency, enhanced drug release under laser irradiation, improved cellular uptake and increased therapeutic effect both in vitro and in vivo. In addition, NIR laser irradiation had a synergistic effect on overcoming MDR. The MDR-overcoming mechanism could be the efficient cellular uptake, enhanced drug release and reduced drug efflux by P-gp. These results demonstrated that ETO@oxCNHs/PEG-PA could be a promising drug delivery system for cancer MDR reversion.

Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the ß-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

A microfluidic linear node array for the study of protein-ligand interactions.

We have developed a microfluidic device for the continuous separation of small molecules from a protein mixture and demonstrated its practical use in the study of protein-ligand binding, a crucial aspect in drug discovery. Our results demonstrated dose-dependent binding between bovine serum albumin (BSA) and its small-molecule site marker, Eosin Y (EY), and found that the binding reached a plateau when the BSA : EY ratio was above 1, which agreed with the eosin binding capacity of BSA reported in literature. By streamline control using a combination of two fundamental building blocks (R and L nodes) with a microdevice operated at a high flow rate (up to 1300 µL h(-1)), a solution barrier was created to "filter" off protein/protein-ligand complexes such that the small unbound molecules were isolated and quantified easily. The percentage decrease of small molecules with increasing protein concentration indicated the presence of binding events. Several fluorophores with different molecular weights were used to test the performance of the microfluidic "filter", which was tunable by 1) the total flow rate, and/or 2) the flow distribution ratio between the two device inlets; both were easily controllable by changing the syringe pump settings. Since the microdevice was operated at a relatively high flow rate, aliquots were easily recovered from the device outlets to facilitate off-chip detection. This microfluidic design is a novel and promising tool for preliminary drug screening.

On-site formation of emulsions by controlled air plugs.

Air plugs are usually undesirable in microfluidic systems because of their detrimental effect on the system's stability and integrity. By controlling the wetting properties as well as the topographical geometry of the microchannel, it is reported herein that air plugs can be generated in pre-defined locations to function as a unique valve, allowing for the on-site formation of various emulsions including single-component droplets, composite droplets with droplet-to-droplet concentration gradient, blood droplets, paired droplets, as well as bubble arrays without the need for precious flow control, a difficult task with conventional droplet microfluidics. Moreover, the self-generated air valve can be readily deactivated (turned off) by the introduction of an oil phase, allowing for the on-demand release of as-formed droplets for downstream applications. It is proposed that the simple, yet versatile nature of this technique can act as an important method for droplet microfluidics and, in particular, is ideal for the development of affordable lab-on-a-chip systems without suffering from scalability and manufacturing challenges that typically confound the conventional droplet microfluidics.

Single layer linear array of microbeads for multiplexed analysis of DNA and proteins.

In this study, a microfluidic platform was developed to generate single layer, linear array of microbeads for multiplexed high-throughput analysis of biomolecules. The microfluidic device is comprised of eight microbead-trapping units, where microbeads were immobilized in a linear array format by the exertion of a negative pressure in the control channel connected to each sieving microstructure. Multiplexed assays were achieved by using a mixture of different spectrally-encoded microbeads functionalized with specific probes, followed by on-chip reaction and detection. The microfluidic-based microbeads array platform was employed for multiplexed analysis of DNA and proteins, as demonstrated by the simultaneous discrimination of four HPV genotypes and the parallel detection of six different proteins. Compared with the off-chip protocols, the on-chip analysis exhibited better reaction efficiency, higher sensitivity and wider linear detection range. Visual inspection and identification of functionalized microbeads were facilitated by the single layer arrangement of microbeads so that accurate data acquisition can be performed during the detection process.