Nanozymatic magnetic nanomixers for enzyme immobilization and multiplexed detection of metabolic disease biomarkers.

Nanozymes with enzyme-mimicking catalytic activity and unique functions have stimulated increasing interest in the biosensing field. Herein, we report a magnetic nanozyme (MNE) with integrated superior peroxidase-like activity and efficient mixing ability. This nanozymatic magnetic nanomixer is synthesized by depositing a Fe2+-doped polydopamine coating on the surface of well-aligned magnetic nanoparticles to form a rigid chain-like nanostructure. Polydopamine coating of the nanozymatic MNE allows for efficient immobilization of natural enzymes such as glucose oxidase, cholesterol oxidase or urate oxidase to produce a series of enzymes-immobilized MNE (MNE@enzymes) with intrinsic multienzyme cascade properties. These MNE@enzymes show synchronously rotating capability in spinning magnetic fields, which leads to an 80∼100% improvement in their overall catalytic efficiencies. In the on-chip detection of small molecular metabolites (i.e., glucose, cholesterol, and uric acid), the rotating MNE@enzymes lead to detection sensitivities 2.1∼4.3 times higher than those of the static ones. Importantly, the consistent performance of the rotating MNE@enzymes offers the possibility of integrating the detection of glucose, free cholesterol and uric acid into a single multiplexing microchip assay with smartphone readout, affording an improved sensitivity, good selectivity and reliability. The designed enzymes-loaded MNEs holds great promise in developing rapid and ultrasensitive measurements of diverse targets of healthcare concerns using portable devices.

Caging Cationic Polymer Brush-Coated Plasmonic Nanostructures for Traceable Selective Antimicrobial Activities.

Cationic polymers are under intense research to achieve prominent antimicrobial activity. However, the cellular and in vivo toxicity caused by nonspecific electrostatic interaction has become a major challenge for their practical applications. Here, the development of a "caging" strategy based on the use of a block copolymer consisting of a stealth block and an anionic block that undergoes degradation in presence of enzymes secreted by selective bacterial pathogens of interest is reported. The results have shown that antimicrobial cationic polymer brushes-coated gold nanorods (AuNRs) can be caged by the block polymer of poly(ethylene glycol) and anionic, lipase-degradable block of ε-caprolactone and methacrylic acid copolymer to afford neutrally charged surfaces. The caged AuNRs are activated by lipase released by bacteria of interest to endow an excellent bactericidal effect but show minimal binding and toxicity against mammalian cells and nonspecific bacteria that do not produce lipase. In this design, AuNRs play multifunctional roles as the scaffolds for polymer brushes, photothermal transducers, and imaging probes for traceable delivery of the activation and delivery of bactericidal cationic polymer brushes. The caging strategy opens new opportunities for the safe delivery of antimicrobial materials for the treatment of bacterial infections.

Multienzyme nanoassemblies: from rational design to biomedical applications.

Multienzyme nanoassemblies (MENAs) that combine the functions of several enzymes into one entity have attracted widespread research interest due to their improved enzymatic performance and great potential for multiple applications. Considerable progress has been made to design and fabricate MENAs in recent years. This review begins with an introduction of the up-to-date strategies in designing MENAs, mainly including substrate channeling, compartmentalization and control of enzyme stoichiometry. The desirable properties that endow MENAs with important applications are also discussed in detail. Then, the recent advances in utilizing MENAs in the biomedical field are reviewed, with a particular focus on biosensing, tumor therapy, antioxidant and drug delivery. Finally, the challenges and perspectives for development of versatile MENAs are summarized.

Magnetic nanochains-based dynamic ELISA for rapid and ultrasensitive detection of acute myocardial infarction biomarkers.

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality globally. The serum levels of a group of cardiac biomarkers have been regarded as important indicators in the routine diagnosis of AMI. The development of rapid, sensitive, and accurate detection methods of AMI biomarkers is urgently needed for the early diagnosis of AMI. Here, a dynamic and pseudo-homogeneous enzyme-linked immunosorbent assay (ELISA) was reported based on the combined use of bioconjugated magnetic nanochains (MNCs) and gold nanoparticles (AuNPs) probes. The capture antibodies-conjugated MNCs served as dynamic nano-mixers to facilitate liquid mixing and as homogeneously dispersed capturing agents to capture and separate specific targets. The AuNPs probes were prepared by co-immobilization of detection antibodies and horseradish peroxidase (HRP) for signals amplification. The design of bioconjugated MNCs and AuNPs probes significantly increased the assay kinetics and improves the assay sensitivity. This novel ELISA strategy realized accurate detection of a panel of AMI biomarkers within 35 min, leading to considerably improved sensitivities compared to that of conventional ELISA method.

Silver nanoprism-based plasmonic ELISA for sensitive detection of fluoroquinolones.

Fluoroquinolones are synthetic antibiotics that are commonly used in animal husbandry, and the consumption of animal products with fluoroquinolone residues has imposed a serious threat to human health. Here, we report a plasmonic enzyme-linked immunosorbent assay (pELISA) method based on oxidative etching of silver nanoprisms (AgNPRs) for the quantitative and qualitative detection of danofloxacin (DAN), a fluoroquinolone antibiotic. AgNPRs that undergo colorimetric changes upon oxidative etching by H2O2 serve as the signal transducer in our design. An indirect competitive pELISA was constructed by introducing biotinylated monoclonal antibody (mAb), streptavidin and biotinylated glucose oxidase, which catalyzes the generation of H2O2 for etching AgNPRs. The quantitative detection limit of the proposed method was 0.24 ng mL-1 for DAN. The qualitative detection limit for DAN reached 0.32 ng mL-1, which was 32-fold lower than that of the assay using 3,3',5,5'-tetramethylbenzidine (TMB) as the signal transducer. The average recoveries of DAN in milk ranged from 103% to 121%, with a coefficient of variation of 0.6-3.41%. The recovery results were further confirmed using liquid chromatography-tandem mass spectrometry. In summary, the proposed AgNPR-etching pELISA exhibits high sensitivity, good accuracy and excellent reliability for the quantitative and qualitative detection of DAN in milk.

A Self-Assembled Plasmonic Substrate for Enhanced Fluorescence Resonance Energy Transfer.

Fluorescence resonance energy transfer (FRET) has found widespread uses in biosensing, molecular imaging, and light harvesting. Plasmonic metal nanostructures offer the possibility of engineering photonic environment of specific fluorophores to enhance the FRET efficiency. However, the potential of plasmonic nanostructures to enable tailored FRET enhancement on planar substrates remains largely unrealized, which are of considerable interest for high-performance on-surface bioassays and photovoltaics. The main challenge lies in the necessitated concurrent control over the spectral properties of plasmonic substrates to match that of fluorophores and the fluorophore-substrate spacing. Here, a self-assembled plasmonic substrate based on polydopamine (PDA)-coated plasmonic nanocrystals is developed to effectively address this challenge. The PDA coating not only drives interfacial self-assembly of the nanocrystals to form closely packed arrays with customized optical properties, but also can serve as a tailored nanoscale spacer between the fluorophores and plasmonic nanocrystals, which collectively lead to optimized fluorescence enhancement. The biocompatible plasmonic substrate that allows convenient bioconjugation imparted by PDA has afforded improved FRET efficiency in DNA microarray assay and FRET imaging of live cells. It is envisioned that the self-assembled plasmonic substrates can be readily integrated into fluorescence-based platforms for diverse biomedical and photoconversion applications.

Synthetic biohybrid peptidoglycan oligomers enable pan-bacteria-specific labeling and imaging: in vitro and in vivo.

Peptidoglycan is the core component of the bacterial cell wall, which makes it an attractive target for the development of bacterial targeting agents. Intercepting its enzymatic assembly with synthetic substrates allows for labeling and engineering of live bacterial cells. Over the past two decades, small-molecule-based labeling agents, such as antibiotics, d-amino acids or monosaccharides have been developed for probing biological processes in bacteria. Herein, peptidoglycan oligomers, substrates for transglycosylation, are prepared for the first time using a top-down approach, which starts from chitosan as a cheap feedstock. A high efficiency of labeling has been observed in all bacterial strains tested using micromolar substrates. In contrast, uptake into mammalian cells was barely observable. Additional mechanistic studies support a hypothesis of bacteria-specific metabolic labeling rather than non-specific binding to the bacterial surface. Eventually, its practicality in bacterial targeting capability is demonstrated in resistant strain detection and in vivo infection models.

Functional Macromolecule-Enabled Colloidal Synthesis: From Nanoparticle Engineering to Multifunctionality.

The synthesis of well-defined inorganic colloidal nanostructures using functional macromolecules is an enabling technology that offers the possibility of fine-tuning the physicochemical properties of nanomaterials and has contributed to a broad range of practical applications. The utilization of functional reactive polymers and their colloidal assemblies leads to a high level of control over structural parameters of inorganic nanoparticles that are not easily accessible by conventional methods based on small-molecule ligands. Recent advances in polymerization techniques for synthetic polymers and newly exploited functions of natural biomacromolecules have opened up new avenues to monodisperse and multifunctional nanostructures consisting of integrated components with distinct chemistries but complementary properties. Here, the evolution of colloidal synthesis of inorganic nanoparticles is revisited. Then, the new developments of colloidal synthesis enabled by functional macromolecules and practical applications associated with the resulting optical, catalytic, and structural properties of colloidal nanostructures are summarized. Finally, a perspective on new and promising pathways to novel colloidal nanostructures built upon the continuous development of polymer chemistry, colloidal science, and nanochemistry is provided.

Lateral Flow Immunoassay Based on Polydopamine-Coated Gold Nanoparticles for the Sensitive Detection of Zearalenone in Maize.

In this work, polydopamine-coated gold nanoparticles (Au@PDAs) were synthesized by the oxidative self-polymerization of dopamine (DA) on the surface of AuNPs and applied for the first time as a signal-amplification label in lateral flow immunoassays (LFIAs) for the sensitive detection of zearalenone (ZEN) in maize. The PDA layer functioned as a linker between AuNPs and anti-ZEN monoclonal antibody (mAb) to form a probe (Au@PDA-mAb). Compared with AuNPs, Au@PDA had excellent color intensity, colloidal stability, and mAb coupling efficiency. The limit of detection of the Au@PDA-based LFIA (Au@PDA-LFIA) was 7.4 pg/mL, which was 10-fold lower than that of the traditional AuNP-based LFIA (AuNP-LFIA) (76.1 pg/mL). The recoveries of Au@PDA-LFIA were 93.80-111.82%, with the coefficient of variation of 1.08-9.04%. In addition, the reliability of Au@PDA-LFIA was further confirmed by the high-performance liquid chromatography method. Overall, our study showed that PDA coating can chemically modify the surface of AuNPs through a simple method and can thus significantly improve the detection sensitivity of LFIA.

Dynamic Magnetic Nanomixers for Improved Microarray Assays by Eliminating Diffusion Limitation.

Microarrays are widely used in high-throughput analysis of DNA, protein, and small molecules. However, the majority of microarray assays need improved assay speed and sensitivity due to the slow molecular diffusion from bulk solutions to probe surfaces. Here, a new class of magnetic nanomixers in DNA and protein microarray assays is reported to eliminate the diffusion constraint through dynamic mixing. It is demonstrated that the dynamic nanomixers can improve the assay kinetics at least by a factor of 4 and 2 for DNA and protein microarray assays, respectively. By using the dynamic nanomixers, the sensitivities of detecting Escherichia coli O157:H7 DNA and prostate specific antigen increase by more than four-fold. The dynamic mixing also greatly reduces the spot-to-spot variation to below 10% across a broad concentration range, providing more accurate assay results. In comparison with existing methods, this magnetic nanomixer-based approach offers rapid turnaround, improved sensitivity, good accuracy, low cost, simple operation, and excellent compatibility with commercial microarrays.

Size-Controllable Magnetic Iron Oxide Nanorods for Biomarker Targeting and Improving Microfluidic Mixing.

Anisotropic nanoparticles, especially gold (Au) nanocubes and nanorods, exhibit unique physical and biological properties compared with their spherical-shaped counterparts, attracting increased attention and effort in developing such a class of nanomaterials for enhanced biomedical applications. Here, we report the dimension-controlled preparation of aqueously stable iron oxide nanorods (IONRs) with tunable dimensions (lengths ranging from 25 to 85 nm and diameters from 5 to 16 nm) and varied saturation magnetization values (from 50 to 79 emu·g-1). Subsequently, the prepared IONRs were evaluated for cell uptake and tested for different biomedical applications that can take advantage of strong magnetic properties of IONRs. In immunomagnetic capturing of biofluidic biomarkers, transferrin-conjugated IONRs demonstrated substantial improvement in efficiency (88%) of capturing transferrin receptor overexpressed pediatric brain tumor medulloblastoma cells (D556) compared with that (47.5%) of commonly used commercial magnetic separation agents, micron-sized Dynabeads. In detecting Aßs and tau proteins, known as Alzheimer's disease biomarkers, antibody-conjugated IONRs showed high sensitivity (91.3%) and specificity (88%). Prepared IONRs also demonstrated rotational movement under the controllable alternating magnetic field. By varying the strength and frequency of an alternating magnetic field, IONRs can be driven as nanoscaled "stirring bars" in the fluid sample in the biofluidic chamber, leading to enhanced liquid mixing for rapid magnetic separations (completed within 106 s).

Magnetic nanochain integrated microfluidic biochips.

Microfluidic biochips hold great potential for liquid analysis in biomedical research and clinical diagnosis. However, the lack of integrated on-chip liquid mixing, bioseparation and signal transduction presents a major challenge in achieving rapid, ultrasensitive bioanalysis in simple microfluidic configurations. Here we report magnetic nanochain integrated microfluidic chip built upon the synergistic functions of the nanochains as nanoscale stir bars for rapid liquid mixing and as capturing agents for specific bioseparation. The use of magnetic nanochains enables a simple planar design of the microchip consisting of flat channels free of common built-in components, such as liquid mixers and surface-anchored sensing elements. The microfluidic assay, using surface-enhanced Raman scattering nanoprobes for signal transduction, allows for streamlined parallel analysis of multiple specimens with greatly improved assay kinetics and delivers ultrasensitive identification and quantification of a panel of cancer protein biomarkers and bacterial species in 1 µl of body fluids within 8 min.

Stable and Biocompatible Mushroom ß-Glucan Modified Gold Nanorods for Cancer Photothermal Therapy.

Naturally occurring ß-glucans have been widely regarded as a natural source for functional foods and pharmaceuticals due to their immunomodulatory property and antitumor activity. However, physicochemically stable and biocompatible ß-glucans are rarely explored as a carrier for nanomaterials to overcome the problems of aggregation and nanotoxicity. Here, we developed highly stable and biocompatible mushroom ß-glucan coated gold nanorods (AuNR-Glu) for cancer photothermal therapy by integrating Pleurotus tuber-regium sclerotial ß-glucan (Glu) and plasmonic gold nanorods (AuNRs) possessing photothermal property in the second near-infrared (NIR-II) window. AuNR-Glu showed high colloidal stability in various biological media, even in simulated gastric fluid. Moreover, AuNR-Glu had low cytotoxicity and high photothermal stability, which are excellent characteristics for photothermal agents for cancer therapy. In vitro experiments showed that AuNR-Glu nanohybrid was effective against MCF-7 (only 4.5 ± 0.9% viability) at a low dose of 20 µg/mL under NIR-II at a safe laser power density (0.75 W/cm2). Natural mushroom ß-glucans are potential functional polymers that can be used to fabricate nanohybrids for biomedical applications.

2D nanomaterials based electrochemical biosensors for cancer diagnosis.

Cancer is a leading cause of death in the world. Increasing evidence has demonstrated that early diagnosis holds the key towards effective treatment outcome. Cancer biomarkers are extensively used in oncology for cancer diagnosis and prognosis. Electrochemical sensors play key roles in current laboratory and clinical analysis of diverse chemical and biological targets. Recent development of functional nanomaterials offers new possibilities of improving the performance of electrochemical sensors. In particular, 2D nanomaterials have stimulated intense research due to their unique array of structural and chemical properties. The 2D materials of interest cover broadly across graphene, graphene derivatives (i.e., graphene oxide and reduced graphene oxide), and graphene-like nanomaterials (i.e., 2D layered transition metal dichalcogenides, graphite carbon nitride and boron nitride nanomaterials). In this review, we summarize recent advances in the synthesis of 2D nanomaterials and their applications in electrochemical biosensing of cancer biomarkers (nucleic acids, proteins and some small molecules), and present a personal perspective on the future direction of this area.

Polydopamine-Enabled Approach toward Tailored Plasmonic Nanogapped Nanoparticles: From Nanogap Engineering to Multifunctionality.

We present a platform strategy that offers diverse flexibility in tailoring the structure and properties of core-shell plasmonic nanoparticles with built-in nanogaps. Our results have demonstrated that polydopamine serves multiple concerted functions as a nanoscale spacer to afford controllable nanogap sizes, a redox-active coating to promote metal shell growth, and a reactive scaffold to exclusively lock molecular probes inside the nanogap for surface-enhanced Raman scattering (SERS). More interestingly, the universal adhesion of polydopamine on diverse colloidal substrates allows for customized synthesis of multishell plasmonic nanogapped nanoparticles (NNPs) and multifunctional hybrid NNPs containing different cores (i.e., magnetic nanoparticles), which are not readily accessible by conventional methods. Internally coupled plasmonic NNPs with broadly tunable spectroscopic properties, highly active SERS, and multifunctionality hold great promise for emerging fields, such as sensing, optoelectronics, and theranostics, as demonstrated by the ultrasensitive SERS detection and efficient photothermal killing of food-borne pathogens here.

Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays.

Label selection is of vital importance for immunochromatographic assays. In this study, the fluorescent microsphere test strip and colloidal gold immunochromatographic test strip (FM-ICTS and CG-ICTS) were developed for the detection of Escherichia coli O157:H7 on the basis of the sandwich format. Two types of labels, namely, colloidal gold particles (CG) and carboxyl-modified fluorescent microspheres (FMs), were compared while coupling with anti-E. coli O157:H7 monoclonal antibody (mAb). The FM-ICTS and CG-ICTS were also compared. Results show that the coupling rate between FMs and mAb was higher than that between CG and mAb. Under optimum conditions, the sensitivity of FM-ICTS was eight times higher than that of CG-ICTS. Approximately 0.1 µg of mAb was used in every FM-ICTS, whereas 0.4 µg of mAb was used in every CG-ICTS. The coefficient of variation of FM-ICTS and CG-ICTS was 4.8% and 16.7%, respectively. The FM-ICTS and CG-ICTS can be stored at room temperature for 12 months and specific to five E. coli O157:H7 strains. Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 10(4) CFU/ml while the CG-ICTS sensitivity was 10(5) CFU/ml. The sensitivity, consumption of antibodies, and coefficient of variation of FM-ICTS were better than those of CG-ICTS for the detection of E. coli O157:H7.