A Hierarchical 3D Nanostructured Microfluidic Device for Sensitive Detection of Pathogenic Bacteria

Efficient capture and rapid detection of pathogenic bacteria from body fluids lead to early diagnostics of bacterial infections and significantly enhance the survival rate. We propose a universal nano/microfluidic device integrated with a 3D nanostructured detection platform for sensitive and quantifiable detection of pathogenic bacteria. Surface characterization of the nanostructured detection platform confirms a uniform distribution of hierarchical 3D nano‐/microisland (NMI) structures with spatial orientation and nanorough protrusions. The hierarchical 3D NMI is the unique characteristic of the integrated device, which enables enhanced capture and quantifiable detection of bacteria via both a probe‐free and immunoaffinity detection method. As a proof of principle, we demonstrate probe‐free capture of pathogenic Escherichia coli (E. coli) and immunocapture of methicillin‐resistant‐Staphylococcus aureus (MRSA). Our device demonstrates a linear range between 50 and 10⁴ CFU mL^?1, with average efficiency of 93% and 85% for probe‐free detection of E. coli and immunoaffinity detection of MRSA, respectively. It is successfully demonstrated that the spatial orientation of 3D NMIs contributes in quantifiable detection of fluorescently labeled bacteria, while the nanorough protrusions contribute in probe‐free capture of bacteria. The ease of fabrication, integration, and implementation can inspire future point‐of‐care devices based on nanomaterial interfaces for sensitive and high‐throughput optical detection.     

Antibody-based Detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Typhimurium Grown in Low-shear Modeled Microgravity

With the advent of prolonged spaceflights, it is important to determine if antibody-based assays can be used to monitor food and water for bacterial contaminants. In the present work, a ground-based high aspect ratio vessel (HARV) was used to determine if low shear modeled microgravity (LSMMG) alters antibody-binding to E. coli O157:H7 and Salmonella enterica serovar Typhimurium. Antibody–bacteria binding was similar under LSMMG and normal gravity because there was no difference in amount of captured bacteria measured by colony forming units (CFU) between assays conducted in the HARV and a conventional roller flask. The ability of E. coli O157:H7 and Salmonella Typhimurium grown in LSMMG to bind specific antibodies was also studied. After incubations of 4, 18 or 36 h in the HARV or a shaking incubator, bacteria were harvested for enzyme-linked immunosorbent assays (ELISA). In the E. coli O157:H7 ELISA using a goat polyclonal primary antibody, LSMMG did not alter the linear range of detection (10⁵–10⁷ cells/ml) nor the signal to noise ratio at any bacterial concentration. Although insignificant changes in signal to noise ratios were evident, LSMMG did not alter the range of detection (10⁵–10⁷ cells/ml) for Salmonella Typhimurium in ELISAs using either a polyclonal or a monoclonal antibody. These results suggest that immunoassays may be used in spacecrafts because LSMMG does not have significant deleterious effects on antibody-binding to bacteria nor does it significantly alter surface antigens necessary for antibody-based methods.     

Surface stress‐induced membrane biosensor based on double‐layer stable gold nanostructures for E. coli detection

The surface stress‐based biosensor has been applied in fast and sensitive identification of Escherichia coli (E. coli)with significance for public health, food, and water safety. However, the stable sensitive element of flexible biosensor based on surface stress is still crucial and challengeable. Here, the authors reported surface stress‐induced biosensors based on double‐layer stable gold nanostructures (D‐AuNS‐SSMB) for E. coli O157:H7 detection. Bacterial detection demonstrates the high stability of the biosensor. The resistance change of biosensor is linear to the logarithmic value of the E. coli O157:H7 concentrations ranging from 10³  to 10⁷  CFU/mL with a limit of detection (LOD) of 43 CFU/mL. The captured signals of D‐AuNS‐SSMB comes from surface stress generated by antigen–antibody binding. In addition, the biosensor exhibits good stability, reproducibility and specificity in detection of E. coli O157:H7 as well. This study provides a new preparation method of stable sensitive element for the E. coli detection.Inspec keywords: microorganisms, biosensors, gold, membranes, stress measurement, nanostructured materials, nanosensorsOther keywords: double‐layer stable gold nanostructures, D‐AuNS‐SSMB, bacterial detection, surface stress‐induced membrane biosensor, escherichia coli detection, water safety, stability, E. coli O157:H7 concentration detection, antigen–antibody binding, Au     

High‐Throughput,Protein‐Targeted Biomolecular Detection Using Frequency‐Domain Faraday Rotation Spectroscopy

A clinically relevant magneto‐optical technique (fd‐FRS, frequency‐domain Faraday rotation spectroscopy) for characterizing proteins using antibody‐functionalized magnetic nanoparticles (MNPs) is demonstrated. This technique distinguishes between the Faraday rotation of the solvent, iron oxide core, and functionalization layers of polyethylene glycol polymers (spacer) and model antibody–antigen complexes (anti‐BSA/BSA, bovine serum albumin). A detection sensitivity of ≈10 pg mL^?1 and broad detection range of 10 pg mL^?1 ? c_BSA ? 100 µ g mL^?1 are observed. Combining this technique with predictive analyte binding models quantifies (within an order of magnitude) the number of active binding sites on functionalized MNPs. Comparative enzyme‐linked immunosorbent assay (ELISA) studies are conducted, reproducing the manufacturer advertised BSA ELISA detection limits from 1 ng mL^?1 ? c_BSA ? 500 ng mL^?1. In addition to the increased sensitivity, broader detection range, and similar specificity, fd‐FRS can be conducted in less than ≈30 min, compared to ≈4 h with ELISA. Thus, fd‐FRS is shown to be a sensitive optical technique with potential to become an efficient diagnostic in the chemical and biomolecular sciences.     

In Vivo Dynamic Monitoring of Bacterial Infection by NIR‐II Fluorescence Imaging

Time window of antibiotic administration is a critical but long‐neglected point in the treatment of bacterial infection, as unnecessary prolonged antibiotics are increasingly causing catastrophic drug‐resistance. Here, a second near‐infrared (NIR‐II) fluorescence imaging strategy based on lead sulfide quantum dots (PbS QDs) is presented to dynamically monitor bacterial infection in vivo in a real‐time manner. The prepared PbS QDs not only provide a low detection limit (10⁴ CFU mL^?1) of four typical bacteria strains in vitro but also show a particularly high labeling efficiency with Escherichia coli (E. coli). The NIR‐II in vivo imaging results reveal that the number of invading bacteria first decreases after post‐injection, then increases from 1 d to 1 week and drop again over time in infected mouse models. Meanwhile, there is a simultaneous variation of dendritic cells, neutrophils, macrophages, and CD8+ T lymphocytes against bacterial infection at the same time points. Notably, the infected mouse self‐heals eventually without antibiotic treatment, as a robust immune system can successfully prevent further health deterioration. The NIR‐II imaging approach enables real‐time monitoring of bacterial infection in vivo, thus facilitating spatiotemporal deciphering of time window for antibiotic treatment.     

Therapeutic Window of Ligand‐Free Silver Nanoparticles in Agar‐Embedded and Colloidal State: In Vitro Bactericidal Effects and Cytotoxicity

The inhibition of bacterial growth through effective non‐toxic antimicrobial substances is of great importance for the prevention and therapy of implant infections in various medical disciplines. For the evaluation of a therapeutic window of silver nanoparticles (AgNPs), their bactericidal properties were tested in agar composites and colloids on four medical relevant bacteria. Therefore, we produced AgNPs using high‐power nanosecond laser ablation in water showing a log‐normal particle diameter distribution centered at 17 nm. Bacteria were incubated with AgNP concentrations ranging from 5 to 70 µg · mL^?1 and the growth rate was recorded. Additionally, cytotoxic effects of AgNPs on human gingival fibroblasts were examined. The experiments demonstrated that laser‐synthesized AgNPs resulted in a significant bacterial growth inhibition of more than 80% at the indicated concentrations in a solid agar model (Pseudomonas aeruginosa 10 µg · mL^?1, Streptococcus salivarius 10 µg · mL^?1, Escherichia coli 20 µg · mL^?1, Staphylococcus aureus 70 µg · mL^?1). In a planktonic bacteria model, the growth of the tested bacteria was significantly delayed by the addition of AgNPs at a concentration of 35 µg · mL^?1. The cytotoxic assays showed limited adverse effects on human fibroblasts at concentrations of less than 20 µg · mL^?1. The present study illustrates the strong antibacterial effects of ligand‐free, laser‐generated AgNPs that exhibit moderate cytotoxic effects, resulting in a therapeutically applicable concentration of AgNPs for medical purposes between 10 and 20 µg · mL^?1.     

Preparation and Characterization of Cellulose Acetate Butyrate/Organoclay Nanocomposites Produced by Extrusion

Nanocomposite films based on cellulose acetate butyrate, modified montmorillonite (Cloisite^® 30B), plasticizer (triethyl citrate) and antimicrobial compounds (carvacrol and cinnamaldehyde) were prepared by extrusion. The effects of the Cloisite^® 30B content and antimicrobial compound types on the morphology of the nanocomposite films were investigated by X‐ray diffraction and transmission electron microscopy. The thermal characteristics of films were analysed by thermogravimetry and differential scanning calorimetry; oxygen and water vapour permeability and tensile strength were determined. The film's antimicrobial behaviour against Listeria innocua, Staphylococcus aureus, Escherichia coli O157:O7 and Saccharomyces cerevisiae was investigated and determined using a viable cell count method. Nanocomposites with a Cloisite^® 30B content of 3 wt% showed better dispersion than nanocomposites with a 5 wt% content. For films with antimicrobial compounds, tensile strength and Young's modulus decreased and water vapour permeability increased (150%) because of the plasticization effect of the antimicrobial compounds (essential oils). The nanocomposites with carvacrol and cinnamaldehyde were effective against the tested Gram‐positive bacteria (reduction of at least 3.0 log CFU/ml) and yeast (reduction of at least 4.0 log CFU/ml). This study demonstrates that antimicrobial cinnamaldehyde and carvacrol can be successfully incorporated into cellulose acetate butyrate nanocomposites and that they have an inhibitory effect against microbial growth in solid medium. It shows the potential use of cellulose acetate butyrate for food packaging applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Sensitive Detection of Carcinoembryonic Antigen Using Stability‐Limited Few‐Layer Black Phosphorus as an Electron Donor and a Reservoir

The instability of few‐layer black phosphorus (FL‐BP) hampers its further applications. Here, it can be demonstrated that the instability of FL‐BP can also be the advantage for application in biosensor. First, gold nanoparticle/FL‐BP (BP‐Au) hybrid is facilely synthesized by mixing Au precursor with FL‐BP. BP‐Au shows outstanding catalytic activity (K = 1120 s^?1 g^?1) and low activation energy (17.53 kJ mol^?1) for reducing 4‐nitrophenol, which is attributed to the electron‐reservoir and electron‐donor properties of FL‐BP, and synergistic interaction of Au nanoparticles and FL‐BP. Oxidation of FL‐BP after catalytic reaction is further confirmed by transmission electron microscope, X‐ray photoelectron spectroscopy, and zeta potentials. Second, the catalytic activity of BP‐Au can be reversibly switched from “inactive” to “active” upon treatment with antibody and antigen in solution, thus providing a versatile platform for label‐free colorimetric detection of biomarkers. The sensor shows a wide detection range (1 pg mL^?1 to –10 µg mL^?1), high sensitivity (0.20 pg mL^?1), and selectivity for detecting carcinoembryonic antigen (CEA). Finally, the biosensor has been used to detect CEA in colon and breast cancer clinical samples with satisfactory results. Therefore, the instability of BP can also be the advantage for application in detecting cancer biomarker in clinic.     

Dielectrophoresis‐Based Protein Enrichment for a Highly Sensitive Immunoassay Using Ag/SiO2 Nanorod Arrays

A nanoscale insulator‐based dielectrophoresis (iDEP) technique is developed for rapid enrichment of proteins and highly sensitive immunoassays. Dense arrays of nanorods (NDs) by oblique angle deposition create a super high electric field gradient of 2.6 × 10²⁴ V² m^?3 and the concomitant strong dielectrophoresis force successfully traps small proteins at a bias as low as 5 V. 1800‐fold enrichment of bovine serum albumin protein at a remarkable rate of up to 180‐fold s^?1 is achieved using oxide coated Ag nanorod arrays with pre‐patterned sawtooth electrodes. Based on this system, an ultrasensitive immunoassay of mouse immunoglobulin G is demonstrated with a reduction in the limit of detection from 5.8 ng mL^?1 (37.6 pM) down to 275.3 fg mL^?1 (1.8 f M), compared with identical assays performed on glass plates. This methodology is also applied to detect a cancer biomarker prostate‐specific antigen spiked in human serum with a detection limit of 2.6 ng mL^?1. This high sensitivity results from rapid biomarker enrichment and metal enhanced fluorescence through the integration of nanostructures. The concentrated proteins also accelerate binding kinetics and enable signal saturation within 1 min. Given the easy fabrication process, this nanoscale iDEP system provides a highly sensitive detection platform for point‐of‐care diagnostics.     

Corona Discharge Plasma Jet Inactivates Food‐borne Pathogens Adsorbed onto Packaging Material Surfaces

The potentiality of corona discharge plasma jet (CDPJ) for disinfection of food packaging materials, including glass, polyethylene, polypropylene, nylon and paper foil, was evaluated. CDPJ was generated using a high voltage (20 kV) pulsed DC power source, at 1.5 A current and 58 kHz frequency. The separation distance between plasma electrode and sample plate during the treatment was 25 mm. Upon treating food pathogens‐loaded packaging materials by the plasma, 4.5–5.0 log/cm² reductions (99.999%) in viable cell counts of Escherichia coli O157:H7 and Staphylococcus aureus were observed in 120 s. Another tested pathogen Salmonella Typhimurium was inactivated by 3.0 log/cm² units. The patterns of inactivation of pathogens are fitted well to Weibull tail model. Compared to untreated controls, the CDPJ‐treated packaging materials exhibited insignificant (p > 0.05) changes in the optical characteristics, tensile strengths, surface temperatures and strain‐induced deformation. Therefore, the most common food packaging materials harboring bacterial pathogens could be disinfected by the CDPJ without compromising physicomechanical properties of materials. Copyright © 2017 John Wiley & Sons, Ltd.     

Synthesis of Multi‐Fullerenes Encapsulated Palladium Nanocage,and Its Application in Electrochemiluminescence Immunosensors for the Detection of Streptococcus suis Serotype 2

A novel functionalized material is synthesized using surface‐decorated fullerene (C₆₀) to encapsulate hollow and porous palladium nanocages (PdNCs), and is applied to fabricate an electrochemiluminescence (ECL) immunosensor for the detection of Streptococcus suis Serotype 2 (SS2). PdNCs with hollow interiors and porous walls are prepared using a galvanic replacement reaction between silver nanocubes and metal precursor salts. Then, C₆₀ reacts with l ‐cysteine (l ‐Cys) to form l ‐Cys functionalized C₆₀ (C₆₀‐l ‐Cys), which has a better biocompatibility, conductivity, and hydrophilicity compared to C₆₀ and possesses abundant –SH groups on the surface. Because of the special interaction between –SH and PdNCs, the obtained C₆₀‐l ‐Cys is adsorbed around the PdNCs to form an interesting structure with multiple spheres encapsulating the cage. The resultant functionalized material (C₆₀‐L‐Cys‐PdNCs) has a high specific surface area, good electrocatalytic ability, and efficient photocatalytic activity, and is used to construct an ECL immunosensor for the detection of SS2. The ECL signal amplified strategy is performed by using the novel coreactant (C₆₀‐l ‐Cys) and in situ generation of O₂ thus creating the S₂O₈^2?‐O₂ ECL system. As a result, a wide linear detection range of 0.1 pg mL^?1 to 100 ng mL^?1 is acquired with a relatively low detection limit of 33.3 fg mL^?1.     

Coculture with Low‐Dose SWCNT Attenuates Bacterial Invasion and Inflammation in Human Enterocyte‐like Caco‐2 Cells

Single walled carbon nanotubes (SWCNTs) have been shown to be highly effective against a wide range of bacteria. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) infection is a well‐known mediator to prolong hospitalization and initiate chronic inflammation, yet the biological effects of SWCNTs on the pathogen‐infected enterocytes remain unclear. Herein, it is shown that the low‐dose SWCNT treatment attenuates the human enterocyte‐like Caco‐2 cells from the damage of E. coli and S. aureus infection by suppressing NLRP3 inflammasome activation. The relatively low‐dose (1 and 10 μg mL^?1) SWCNT treatments reduce the adhesion and invasion of E. coli and S. aureus to Caco‐2 cells, increase the cell viability and proliferation, reduce the tight junction permeability, and restitute the integrity of cell surface microvilli structure, meanwhile has low cytotoxicity to the host cells. The low‐dose SWCNT treatment further reduces the NLRP3‐mediated IL‐1β secretion in the infected cells. The results identify that a low‐dose SWCNT treatment serves a protective function for the E. coli‐ and S. aureus‐infected Caco‐2 cells by negatively regulating mitochondrial reactive oxygen species‐mediated NLRP3 inflammasome activation.     

Green Fabrication of Amphiphilic Quaternized β‐Chitin Derivatives with Excellent Biocompatibility and Antibacterial Activities for Wound Healing

Bacterial infection has always been a great threat to public health, and new antimicrobials to combat it are urgently needed. Here, a series of quaternized β‐chitin derivatives is prepared simply and homogeneously in an aqueous KOH/urea solution, which is a high‐efficiency, energy‐saving, and “green” route for the modification of chitin. The mild reaction conditions keep the acetamido groups of β‐chitin intact and introduce quaternary ammonium groups on the primary hydroxyl at the C‐6 position of the chitin backbone, allowing the quaternized β‐chitin derivatives (QCs) to easily form micelles. These QCs are found to exhibit excellent antimicrobial activities against Escherichia coli, Staphylococcus aureus, Candida albicans, and Rhizopus oryzae with minimum inhibitory concentrations (MICs) of 8, 12, 60, and 40 µg mL^?1, respectively. As a specific highlight, their inherent outstanding biocompatibility and significant accelerating effects on the healing of uninfected, E. coli‐infected, and S. aureus‐infected wounds imply that these novel polysaccharide‐based materials can be used as dressings for clinical skin regeneration, particularly for infected wounds.     

Regenerable antimicrobial silica gel with quaternarized N-halamine

A novel regenerable antimicrobial silica gel was prepared by a dehydration between silanols of silica gel and hydrolyzed 3-chloropropyltrimethoxysilane, a quaternization between grafted hydrolyzed 3-chloropropyltrimethoxysilane and (5,5-dimethylhydantoinyl-3-ylethyl)-dimethylamine (DHEDA), and a chlorination of amide groups of DHEDA. The as-prepared antimicrobial silica gel was characterized by FT-IR and X-ray photoelectron spectroscopy. Antimicrobial tests showed that the as-prepared antimicrobial silica gel was capable of about a 6-log inactivation of Staphylococcus aureus and Escherichia coli O157:H7 within 10 min of contact. Interestingly, more than 2 × 10⁴ CFU/mL of Escherichia coli O157:H7 could be almost inactivated under flowing water condition. Furthermore, the as-prepared antimicrobial silica gel exhibits good regenerability and storage stability.     

Highly Sensitive Detection of Protein Biomarkers with Organic Electrochemical Transistors

The analysis of protein biomarkers is of great importance in the diagnosis of diseases. Although many convenient and low‐cost electrochemical approaches have been extensively investigated, they are not sensitive enough in the detection of protein biomarkers with low concentrations in physiological environments. Here, this study reports a novel organic‐electrochemical‐transistor‐based biosensor that can successfully detect cancer protein biomarkers with ultrahigh sensitivity. The devices are operated by detecting electrochemical activity on gate electrodes, which is dependent on the concentrations of proteins labeled with catalytic nanoprobes. The protein sensors can specifically detect a cancer biomarker, human epidermal growth factor receptor 2, down to the concentration of 10^?14 g mL^?1, which is several orders of magnitude lower than the detection limits of previously reported electrochemical approaches. Moreover, the devices can successfully differentiate breast cancer cells from normal cells at various concentrations. The ultrahigh sensitivity of the protein sensors is attributed to the inherent amplification function of the organic electrochemical transistors. This work paves a way for developing highly sensitive and low‐cost biosensors for the detection of various protein biomarkers in clinical analysis in the future.     

Hyperboloid‐Drum Microdisk Laser Biosensors for Ultrasensitive Detection of Human IgG

Whispering gallery mode (WGM) microresonators have been used as optical sensors in fundamental research and practical applications. The majority of WGM sensors are passive resonators that require complex systems, thereby limiting their practicality. Active resonators enable the remote excitation and collection of WGM‐modulated fluorescence spectra, without requiring complex systems, and can be used as alternatives to passive microresonators. This paper demonstrates an active microresonator, which is a microdisk laser in a hyperboloid‐drum (HD) shape. The HD microdisk lasers are a combination of a rhodamine B‐doped photoresist and a silica microdisk. These HD microdisk lasers can be utilized for the detection of label‐free biomolecules. The biomolecule concentration can be as low as 1 ag mL^?1, whereas the theoretical detection limit of the biosensor for human IgG in phosphate buffer saline is 9 ag mL^?1 (0.06 a_M). Additionally, the biosensors are able to detect biomolecules in an artificial serum, with a theoretical detection limit of 9 ag mL^?1 (0.06 a_M). These results are approximately four orders of magnitude more sensitive than those for the typical active WGM biosensors. The proposed HD microdisk laser biosensors show enormous detection potential for biomarkers in protein secretions or body fluids.     

Preparation and characterization of antibacterial graphene oxide functionalized with polymeric <Emphasis Type="Italic">N</Emphasis>-halamine

Organic–inorganic composites have also gained much attention owing to their excellent combined properties. For the enhancement of the bacteria-inactivation ability of graphene oxide (GO), poly[5,5-dimethyl-3-(3′-triethoxysilylpropyl)hydantoin] (PSPH) was synthesized and attached onto GO through covalent bond. The synthesized inorganic–organic composites (GO-PSPH) were characterized by FT-IR, XPS, XRD, AFM, SEM, etc. After chlorination treatment by sodium hypochlorite, biocidal efficacies of the chlorinated GO-PSPH (GO-PSPH-Cl) against S. aureus (ATCC 6538) and E. coli O157:H7 (ATCC 43895) were tested. The antibacterial testing results showed that the GO-PSPH-Cl has great antibacterial activity and could completely inactivate 5.5 × 10⁶ CFU/mL of S. aureus and 1.2 × 10⁸ CFU/mL of E. coli O157:H7 within 30 and 10 min of contact time, respectively.     

Rapid and Digital Detection of Inflammatory Biomarkers Enabled by a Novel Portable Nanoplasmonic Imager

New point‐of‐care diagnostic devices are urgently needed for rapid and accurate diagnosis, particularly in the management of life‐threatening infections and sepsis, where immediate treatment is key. Sepsis is a critical condition caused by systemic response to infection, with chances of survival drastically decreasing every hour. A novel portable biosensor based on nanoparticle‐enhanced digital plasmonic imaging is reported for rapid and sensitive detection of two sepsis‐related inflammatory biomarkers, procalcitonin (PCT) and C‐reactive protein (CRP) directly from blood serum. The device achieves outstanding limit of detection of 21.3 pg mL^?1 for PCT and 36 pg mL^?1 for CRP, and dynamic range of at least three orders of magnitude. The portable device is deployed at Vall d'Hebron University Hospital in Spain and tested with a wide range of patient samples with sepsis, noninfectious systemic inflammatory response syndrome (SIRS), and healthy subjects. The results are validated against ultimate clinical diagnosis and currently used immunoassays, and show that the device provides accurate and robust performance equivalent to gold‐standard laboratory tests. Importantly, the plasmonic imager can enable identification of PCT levels typical of sepsis and SIRS patients in less than 15 min. The compact and low‐cost device is a promising solution for assisting rapid and accurate on‐site sepsis diagnosis.