Guanidinium-Functionalized Polymer Dielectrics for Triboelectric Bacterial Detection.

Development of rapid detection strategies that target potentially pathogenic bacteria has gained increasing attention due to the increasing awareness for better health and safety. In this study, we evaluate an intrinsically antimicrobial polymer, 2Gdm, which is a poly(norbornene)-based functional polymer featuring guanidinium groups as side chains, for bacterial detection by the means of triboelectric nanogenerators (TENGs) and triboelectric nanosensors (TENSs). Attachment of bacteria to the sensing layer is anticipated to alter the overall triboelectric properties of the underlying polymer layer. The positively charged guanidinium functional groups can interact with the negatively charged phospholipid bilayer of bacteria and lead to bacterial death, which can then be detected by optical microscopy, X-ray photoelectron microscopy, and more advanced self-powered sensing techniques such as TENGs and TENSs. The double bonds present along the poly(norbornene) backbone allow for thermally induced cross-linking to obtain X-2Gdm and thus rendering materials remain stable in water. By monitoring the change in voltage output after immersion in various concentrations of Gram-negative Escherichia coli (E. coli) and Gram-positive Streptococcus pneumoniae (S. pneumoniae), we have demonstrated the utility of X-2Gdm as a new polymer dielectric for autonomous bacterial detection. As the bacterial concentration increases, the amount of adsorbed bacteria also increases, resulting in a decrease in the surface potential of the X-2Gdm thin film; this reduction in surface potential can cause a decrease in the triboelectric output for both TENGs and TENSs, which serves as a key working mechanism for facile bacterial detection. TENG and TENS systems are capable of detecting E. coli and S. pneumoniae within a range of 4 × 105 to 4 × 108 CFU/mL with a limit of detection of 106 CFU/mL. This report highlights the promising prospects of employing TENGs and TENSs as innovative sensing technologies for rapid bacterial detection by leveraging the electrostatic interactions between bacterial cell membranes and cationic groups present on polymer surfaces.

Dual and Sequential Locked/Unlocked Photochromic Effects on FRET Controlled Singlet Oxygen Processes by Contracted/Extended Forms of Diarylethene-Based [1]Rotaxane Nanoparticles.

Manipulations of singlet oxygen (1 O2 ) generations by the integration of both aggregation-induced emission luminogen (AIEgen) photosensitizer and photochromic moieties have diversified features in photodynamic therapy applications. Through Förster resonance energy transfer (FRET) pathway to induce red PL emissions (at 595 nm) for 1 O2 productions, [1]rotaxane containing photosensitive tetraphenylethylene (TPE) donor and photochromic diarylethene (DAE) acceptor is introduced to achieve dual and sequential locked/unlocked photoswitching effects by pH-controlled shuttling of its contracted/extended forms. Interestingly, the UV-enabled DAE ring closure speeds follow the reversed trend of DAE self-constraint degree as: contracted < extended < noninterlocked forms in [1]rotaxane analogues, thus FRET processes can be adjusted in contracted/extended forms of [1]rotaxane upon UV irradiations. Accordingly, the contracted form of [1]rotaxane is FRET-OFF locked and inert to UV exposure due to the larger bending conformation of DAE parallel (p-)conformer, compared with its extended and noninterlocked analogues possessing switchable FRET-OFF/ON behaviors activated by dual and sequential pH- and photoswitching. Owing to the advantages of 1 O2 productions tuned by multistimuli inputs (pH, UV, and blue light), an useful logic circuit for toxicity outputs of the surface modified [1]rotaxane nanoparticles (NPs) has been demonstrated to offer promising 1 O2 productions and managements based on mechanically interlocked molecules for future bioapplications.

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (K D = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

Integration of Ni/NiO nanoparticles and a microfluidic ELISA chip to generate a sensing platform for Streptococcus pneumoniae detection.

Enzyme-linked immunosorbent assays (ELISAs) are tests that uses antibody recognition and enzyme catalytic activity to identify a substance, and they have been widely used as a diagnostic tool in the clinic. However, performing an ELISA requires various liquid handling steps and long binding times. To solve this problem, we developed a magnetic microfluidic ELISA system (MMF-ELISA). Integration with nickel magnetic nanoparticles can streamline the ELISA process in a fully automated manner for Streptococcus pneumoniae detection. First, we synthesized paramagnetic surface-oxidized nickel nanoparticles (Ni/NiO NPs) to carry protein G. Then, we assembled a SUM290 (UlaG)-specific antibody on protein G. Finally, we integrated the NPs on a microfluidics chip for S. pneumoniae detection. The chip contains three different layers to trap the solutions; the bottom layer SiO2 is patterned on hydrophobic polymers and integrated with the middle layer PDMS and the top layer PMMA. With Arduino and motor IC, we developed an automated platform for S. pneumoniae detection. Microfluidic ELISAs can reduce the manual handling and operation time. Furthermore, the developed system can be extended to multiple areas for ELISA-related assays. This economical, rapid and portable system may become a promising platform for sensing S. pneumoniae in clinical applications.

Activation of apoptosis by Salmonella pathogenicity island-1 effectors through both intrinsic and extrinsic pathways in Salmonella-infected macrophages.

BACKGROUND: Salmonella enterica serovar Typhimurium, a non-typhoidal food-borne pathogen, causes acute enterocolitis, bacteremia, extraintestinal focal infections in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) contribute to invading into host cellular cytosol, residing in Salmonella-containing vacuoles for intracellular survival, and inducing cellular apoptosis. This study aimed to better understand the mechanism underlying apoptosis in Salmonella-infected macrophages. METHODS: S. Typhimurium SL1344 was used to evaluate extrinsic and intrinsic apoptosis pathways in THP-1 monocyte-derived macrophages in response to Salmonella infection. RESULTS: Activated caspase-3-induced apoptosis pathways, including extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, in Salmonella-infected macrophages were verified. THP-1 cells with dysfunction of TLR-4 and TLR-5 and Salmonella SPI-1 and SPI-2 mutants were constructed to identify the roles of the genes associated with programmed cell death in the macrophages. Caspase-3 activation in THP-1 macrophages was induced by Salmonella through TLR-4 and TLR-5 signaling pathways. We also identified that SPI-1 structure protein PrgH and effectors SipB and SipD, but not SPI-2 structure protein SsaV, could induce apoptosis via caspase-3 activation and reduce the secretion of inflammation marker TNF-α in the Salmonella-infected cells. The two effectors also reduced the translocation of the p65 subunit of NF-κB into the nucleus and the expression of TNF-α, and then inflammation was diminished. CONCLUSION: Non-typhoid Salmonella induced apoptosis of macrophages and thereby reduced inflammatory cytokine production through the expression of SPI-1. This mechanism in host-pathogen interaction may explain why Salmonella usually manifests as occult bacteremia with less systemic inflammatory response syndrome in the bloodstream infection of children.

Efficient FRET Approaches toward Copper(II) and Cyanide Detections via Host-Guest Interactions of Photo-Switchable [2]Pseudo-Rotaxane Polymers Containing Naphthalimide and Merocyanine Moieties.

A supramolecular [2]pseudo-rotaxane containing a naphthalimide-based pillararene host and a spiropyran-based imidazole guest was synthesized and investigated in a semiaqueous solution with 90% water fraction. Upon UV exposure, the close-form structure of nonemissive spiropyran guest could be transformed into the open-form structure of red-emissive merocyanine guest reversibly, which was utilized as a monofluorophoric sensor to detect copper(II) and cyanide ions. Moreover, the naphthalimide host as an energy donor with green photoluminescence (PL) emission at 505 nm was complexed with the merocyanine guest as an energy acceptor with red PL emission at 650 nm in 1:1 molar ratio to generate a [2]pseudo-rotaxane polymer, which was further verified by the diffusion coefficients of DOSY nuclear magnetic resonance (NMR) measurements. Due to the Förster resonance energy transfer (FRET) processes, the bifluorophoric [2]pseudo-rotaxane produced more efficient ratiometric PL behavior to induce a stronger red PL emission than that of the monofluorophoric guest; therefore, the PL sensor responses of the supramolecular [2]pseudo-rotaxane toward copper(II) and cyanide ions could be further amplified via the FRET-OFF processes to turn off red PL emission of the reacted merocyanine acceptor and to recover green PL emission of the naphthalimide donor. Accordingly, the best and prominent values of the limit of detection (LOD) for the host-guest detections toward Cu2+ and CN- were 0.53 and 1.34 µM, respectively. The highest red MC emission with the optimum FRET processes of [2]pseudo-rotaxane was maintained around room temperature (20-40 °C) in wide pH conditions (pH = 3-13), which can be utilized in the cell viability tests to prove the nontoxic and remarkable biomarker of [2]pseudo-rotaxane to detect Cu2+ and CN- in living cells. The developed FRET-OFF processes with ratiometric PL behavior of the bifluorophoric supramolecular [2]pseudo-rotaxane polymer will open a new avenue to the future applications of chemo- and biosensors.

Snake Tracks in Polymer Land: Wavy Polymer Structures via Selective Solvent Vapor Annealing.

Wavy patterns are interesting geometric patterns and commonly seen in nature, such as serpentine streams or snake tracks in the sand. Although many efforts have been devoted to fabricating artificial wavy structures, it remains a great challenge to obtain wavy structures with controllable curvatures and desired functional properties. Here, we present an unprecedented approach to generate wavy polymer structures by annealing electrospun core-shell fibers on polymer films. Polystyrene (PS)/poly(methyl methacrylate) (PMMA) core-shell fibers, produced via the viscosity-induced phase separation in the electrospinning process, are annealed on PMMA films using vapors of acetic acid, a selective solvent for PMMA but not for PS. After the swollen PMMA chains of the PMMA shells are shed, the revealed PS cores start to buckle, driven by the elastic force from the strain release, forming the wavy structures. The degrees of the buckling, measured by the curvatures and the amplitudes of the wavy structures, are controlled by the annealing times. Furthermore, fluorescent properties are selectively introduced to the wavy structures using pyrene solutions or pyrene-containing vapors, demonstrating the potential application as fluorescent wavy materials.

An antifouling peptide-based biosensor for determination of Streptococcus pneumonia markers in human serum.

We report a peptide-based sensor that involves a multivalent interaction with L-ascorbate 6-phosphate lactonase (UlaG), a protein marker of Streptococcus pneumonia. By integrating the antifouling feature of the sensor, we significantly improved the signal-to-noise ratio of UlaG detection. The antifouling surface was fabricated via electrodeposition using an equivalent mixture of 4-amino-N,N,N-trimethylanilinium and 4-aminobenzenesulfonate. This antifouling layer not only effectively reduces the non-specific adsorption on the biosensor but also decreases the charge transfer resistance (Rct) of the screen-printed carbon electrode. The aniline-modified S7 peptide, an UlaG-binding peptide, was pre-synthesized and further electrochemically modified to bind onto the antifouling layer. Bio-electrochemical analysis confirms that the antifouling S7-peptide sensor binds strongly to the UlaG with a dissociation constant (Kd) = 0.5 nM. This strong interaction can be attributed to a multivalent interaction between the biosensor and the heximeric form of UlaG. To demonstrate the potential for clinical application, further detection of Streptococcus pneumonia from 50 to 5×104 CFU/mL were successfully performed in 25% human serum.

Development of a novel engineered antibody targeting Neisseria species.

OBJECTIVES: A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv). RESULTS: Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine. CONCLUSIONS: The recombinant scFv could detect Neisseria strains at 106 CFU/ml.