General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers.

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

Electrochemiluminescence for Electric-Driven Antibacterial Therapeutics.

The employment of physical light sources in clinical photodynamic therapy (PDT) system endows it with a crucial defect in the treatment of deeper tissue lesions due to the limited penetration depth of light in biological tissues. In this work, we constructed for the first time an electric driven luminous system based on electrochemiluminescence (ECL) for killing pathogenic bacteria, where ECL is used for the excitation of photosensitizer instead of a physical light source to produce reactive oxygen species (ROS). We named this new strategy as ECL-therapeutics. The mechanism for the ECL-therapeutics is dependent on the perfect spectral overlap and energy transfer from the ECL generated by luminol to photosensitizer, cationic oligo(p-phenylenevinylene) (OPV), to sensitize the surrounding oxygen molecule into ROS. Furthermore, taking into account the practical application of our ECL-therapeutics, we used flexible hydrogel to replace the liquid system to develop hydrogel antibacterial device. Because the chemical reaction is a slow process in the hydrogel, the luminescence could last for more than 10 min after only electrifying for five seconds. This unique persistent luminescence characteristic with long afterglow life makes them suitable for persistent antibacterial applications. Thus, stretchable and persistent hydrogel devices are designed by integrating stretchable hydrogel, persistent ECL and antibacterial function into hydrogel matrices. This novel strategy avoids the employment of external light source, making it simple, convenient and controllable, which exploits a new field for ECL beyond sensors and also opens up a new model for PDT.

Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells.

Bacteria quorum sensing (QS) has attracted significant interest for understanding cell-cell communication and regulating biological functions. In this work, we demonstrate that water-soluble cationic conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. With bacteria coated in the aggregate, PFP-G2 can induce the bacteria QS system and prolong the time duration of QS signal molecules (autoinducer-2 (AI-2)) production. The prolonged AI-2 can bind with specific protein and continuously regulate downstream gene expression. Consequently, the bacteria show a higher survival rate against antibiotics, resulting in decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in E. coli. This method can be used to understand cell-cell communication and regulate biological functions, such as the production of signaling molecules, antibiotics, other microbial metabolites, and even virulence.

Enhanced bidirectional extracellular electron transfer based on biointerface interaction of conjugated polymers-bacteria biohybrid system.

The low bacteria loading capacity and low extracellular electron transfer (EET) efficiency are two major bottlenecks restricting the performance of the bioelectrochemical systems from practical applications. Herein, we demonstrated that conjugated polymers (CPs) could enhance the bidirectional EET efficiency through the intimate biointerface interactions of CPs-bacteria biohybrid system. Upon the formation of CPs/bacteria biohybrid, thick and intact CPs-biofilm formed which ensured close biointerface interactions between bacteria-to-bacteria and bacteria-to-electrode. CPs could promote the transmembrane electron transfer through intercalating into the cell membrane of bacteria. Utilizing the CPs-biofilm biohybrid electrode as anode in microbial fuel cell (MFC), the power generation and lifetime of MFC had greatly improved based on accelerated outward EET. Moreover, using the CPs-biofilm biohybrid electrode as cathode in electrochemical cell, the current density was increased due to the enhanced inward EET. Therefore, the intimate biointerface interaction between CPs and bacteria greatly enhanced the bidirectional EET, indicating that CPs exhibit promising applications in both MFC and microbial electrosynthesis.

Manipulating the monomer-dimer transformation of a heptamethine cyanine ligand: near infrared chromogenic recognition of biothiols.

A novel absorbance recovery method has been developed for the determination of biothiols with a near-infrared reagent. This method employs a two-reagent system composed of cation heptamethine cyanine (CyL) and Hg2+. The absorbance of CyL, with a maximum peak at 760 nm, was decreased due to addition of Hg2+, but recovered when biothiols were added. Under optimal conditions, the reciprocal extent of recovered absorbance was proportional to the concentration of biothiols. The calibration curves are linear over the range of (0.3-7.0) × 10-6 M for cysteine, (1.0-10.0) × 10-6 M for homocysteine and (1.0-9.0) × 10-6 M for glutathione. Because of the specific affinity of Hg2+for biothiols, there is minimal interference from other amino acids. This method has been successfully applied to the determination of homocysteine in human urine samples with satisfactory results.

A Label-Free, Mix-and-Detect ssDNA-Binding Assay Based on Cationic Conjugated Polymers.

The accurate, simple, and efficient measurement of the concentration of single-stranded DNA (ssDNA) is important for many analytical applications, such as DNA adsorption, biosensor design, and disease diagnosis, but it is still a challenge. Herein, we studied a cationic conjugated polymer (CCP)-based ssDNA assay taking advantage of the obvious fluorescence change of CCPs upon binding ssDNA. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) achieved an apparent dissociation constant (Kd) of 57 ± 4 nM for ssDNA, indicating a very high binding affinity between PMNT and ssDNA. This allowed us to develop a CCP-based ssDNA biosensor with a detection limit of 0.6 nM, similar to the fluorescence-dye-based method using SYBR Green I and SYBR Gold. Our CCP-based biosensor produced smaller differences among ssDNA samples with different base compositions. In addition, the existence of double-stranded DNA (dsDNA) at different concentrations did not interfere with the fluorescence of PMNT, indicating that our CCP-based biosensor was more suitable for the measurement of ssDNA. Compared with fluorescence-intensity-based quantification, our CCP system allowed ratiometric quantification, which made the calibration easier and more robust. We then applied our method to the quantification of ssDNA on AuNPs using both unmodified and thiolated ssDNA, and the accurate quantification of ssDNA was achieved without any fluorophore modification. This method provides an alternative approach for the measurement of ssDNA.

Controlling the Interaction between Fluorescent Gold Nanoclusters and Biointerfaces for Rapid Discrimination of Fungal Pathogens.

Nondestructive detection and discrimination of fungal pathogens is essential for rapid and precise treatment, which further effectively prevents antifungal resistance from overused drugs. In this work, fluorescent gold nanoclusters served as the basis for discriminating Candida species. Varied on surface ligands, these gold nanoclusters demonstrated different optical properties as a result of the perturbation effects of ligands. The biointerface interaction between the surface ligands of gold nanoclusters and the cell walls of Candida species can be constructed, and their restriction on ligands perturbation effect produced enhanced fluorescence signals. Owing to the variation of the cell wall composition, cells of different Candida species demonstrated different degrees of association with the gold nanoclusters, leading to discriminable amounts of fluorescence enhancements. The reverse signal response from these gold nanoclusters gives rise to a synergistic and effective assay that allows identification of Candida species.

Neural Stem Cell-Laden Self-Healing Polysaccharide Hydrogel Transplantation Promotes Neurogenesis and Functional Recovery after Cerebral Ischemia in Rats.

Exploring a strategy to effectively repair cerebral ischemic injury is a critical requirement for neuroregeneration. Herein, we transplanted a neural stem cell (NSC)-laden self-healing and injectable hydrogel into the brains of ischemic rats and evaluated its therapeutic effects. We observed an improvement in neurological functions in rats transplanted with the NSC-laden hydrogel. This strategy is sufficiently efficient to support neuroregeneration evidenced by NSC proliferation, differentiation, and athletic movement recovery of rats. This therapeutic effect relates to the inhibition of the astrocyte reaction and the increased expression of vascular endothelial growth factor. This work provides a novel approach to repair cerebral ischemic injury.

Cationic conjugated polymers for enhancing beneficial bacteria adhesion and biofilm formation in gut microbiota.

It is important to develop efficient therapeutic methods to maintain a healthy balance among gut microbiota by increasing the beneficial bacteria and decreasing the harmful bacteria. In this work, a cationic polythiophene derivative poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) with quaternary ammonium groups as side chains has been used for efficiently promoting the initial adhesion and biofilm formation of beneficial bacteria in gut microbiota. Upon addition of PMNT, three species of gut microbiota have an increased biofilm formation ability (216.5 % for Escherichia coli (E. coli), 130.7 % for Bifidobacterium infantis (B. infants) and 47.6 % for Enterococcus faecalis (E. faecalis)). As the initial adhesion of bacteria to a surface is an essential step during biofilm formation, PMNT can promote the attachment of bacteria by forming bacteria /PMNT aggregates which possess more cell-to-cell interactions. RNA sequencing results of bacteria within biofilm indicate that the utilization of carbohydrate and glycan is accelerated in the presence of PMNT, leading to enhanced quorum sensing and biofilm formation of E. coli. After forming biofilm, beneficial bacteria have an enhanced resistance to adverse environmental conditions which is significant for maintaining the balance of gut microbiota. Conjugated polymers exhibit a good potential application in modulating the balance of gut microbiota and development of new probiotics drugs.

Conjugated Polymer-Quantum Dot Hybrid Materials for Pathogen Discrimination and Disinfection.

In this work, a new platform for pathogen discrimination and killing based on a conjugated polymer-quantum dot hybrid material was designed and constructed through the fluorescence resonance energy transfer (FRET) process. The hybrid material comprises water-soluble anionic CdSe/ZnS quantum dots (QDs) and a cationic poly(fluorene-alt-phenylene) derivative (PFP) through electrostatic interactions, thus promoting efficient FRET between PFP and QDs. Upon addition of different pathogen strains, the FRET from PFP to QDs was interrupted because of the competitive binding between PFP and the pathogens. Complexation of PFP and QDs also reduced the dark toxicity to a more desirable level, therefore potentially realizing the controllable killing of pathogens. The technique provides a promising theranostic platform in pathogen discrimination and disinfection based on FRET and phototoxicity of the PFP and QDs.

Conjugated Polymer Enhanced Photoelectric Response of Self-Circulating Photosynthetic Bioelectrochemical Cell.

A water-oxygen-water photosynthetic bioelectrochemical cell (PBEC) comprising hybrid poly(fluorene-alt-phenylene) (PFP)/PSII-enriched membranes (BBY) photoanode and bilirubin oxidase (BOD) biocathode has been designed and fabricated. In the PBEC, water is split into oxygen, protons, and electrons through light-dependent reaction of PSII at the photoanode, and oxygen is converted into water catalyzed by BOD at the biocathode, forming the electronic circuit and generating current. At the photoanode, PFP can simultaneously accelerate the photosynthetic water oxidation and the electron transfer between BBY and electrode. Interestingly, the photocurrent density produced by PBEC after the introduction of PFP reaches 1.05 ± 0.01 µA/cm2, which is 2.5 times more than that of the BBY electrode, indicating that conjugated polymer can enhance the photoelectric response of PBEC.

Photoelectrochemical Strategy for Discrimination of Microbial Pathogens Using Conjugated Polymers.

A photoelectrochemical (PEC) biosensor for facile and sensitive identification of pathogenic microorganisms was developed. Cationic poly(phenylene vinylene) derivative (PPV) as photoelectrochemical active species was modified on the electrode. Under light irradiation, PPV could be excited and generate efficient photocurrent. PPV also had the ability to bind with negatively charged membrane of pathogenic microorganisms, which hindered the electron transfer between electrode and electrolyte. As a result, the photocurrent would decrease obviously. For E. coli, B. subtilis and C. albicans, the photocurrent density was reduced by 18, 33 and 59 %, respectively. Based on the reduction degree of the photocurrent after capturing different types of species of pathogenic microorganisms, a PEC sensor for discrimination of pathogenic microorganisms was realized.

Photothermal-Responsive Conjugated Polymer Nanoparticles for Remote Control of Gene Expression in Living Cells.

Remote control and noninvasive manipulation of cellular bioprocess has received intensive attention as a powerful technology to control cell functions. Here, a strategy is developed to remotely control intracellular gene expression with high spatial and temporal resolutions by using photothermal-responsive conjugated polymer nanoparticles (CPNs) as the transducer under near-infrared light irradiation. After being modified with positive charged peptide, the CPNs with superior photothermal conversion capacity could effectively coat on the surface of living cells and generate localized heat to trigger target gene expression. The heat-inducible heat shock protein-70 promoter starts transcription of downstream EGFP gene in response to heat shock, thus producing green fluorescent protein in the living cells. The combination of heat-inducible gene promoter and photothermal-responsive CPNs provides a method for the development of thermogenetics.

Biofilm Inhibition and Elimination Regulated by Cationic Conjugated Polymers.

In this work, we demonstrate that water-soluble conjugated polymers (PFP) have the ability to inhibit biofilm formation and eradicate mature established biofilm using reactive oxygen species (ROS) produced by PFP under white light irradiation. Upon addition of PFP to planktonic Staphylococcus aureus (S. aureus), electrostatic interactions bring cationic PFP to the surface of S. aureus, which possesses negative charges. As the amount of PFP coated on S. aureus becomes saturated, the interactions of bacteria to bacteria and bacteria to surface may be disrupted, resulting in reduced biofilm formation. After the biofilm matures, those PFP on the surface of the biofilm can generate ROS under white light irradiation, which has the ability to inactivate bacteria nearby. Once the biofilm is broken, PFP can penetrate throughthe biofilm and continuously generate ROS under irradiation, resulting in biofilm disruption. As a consequence, this makes conjugated polymers a very promising material for the disruption of biofilm in biomedical and industrial applications.

Cationic Poly(p-phenylene vinylene) Materials as a Multifunctional Platform for Light-Enhanced siRNA Delivery.

In this work, a multifunctional platform based on the versatile function of cationic poly(p-phenylene vinylene) (PPV) derivative has been developed for white light-enhanced siRNA delivery in a remote control manner. Conjugated polyelectrolyte PPV, which possesses cationic and amphipathic features, can effectively self-assemble with siRNA and deliver siRNA into living cells. More importantly, PPV can sensitize surrounding oxygen into reactive oxygen species (ROS) upon exposure to white light and disrupt the endosomal membrane, and the resulting enhanced endosomal escape increases the gene silencing ability of siRNA. Besides, due to high fluorescent emission, PPV can also be used to image the siRNA delivery and intracelluar location. Therefore, by taking full advantage of PPV, this strategy provides a new enhanced siRNA delivery in a non-invasive and spatiotemporal photochemical manner.

An optical nanoruler based on a conjugated polymer-silver nanoprism pair for label-free protein detection.

An optical nanoruler system based on a conjugated polyelectrolyte-silver nanoprism pair is developed for label-free protein detection by taking advantage of the metal-enhanced fluorescence effect of silver nanostructures. Antibody-antigen interactions induce a change in the metal-fluorophore distance, followed by the response of a fluorescent signal of the conjugated polyelectrolyte. The system is used to detect target antigens sensitively and selectively.

Low concentration of serum helps to maintain the characteristics of NSCs/NPCs on alkali-treated PHBHHx film in vitro.

OBJECTIVE: Efforts have been made by tissue engineers to create a permissive environment for neural regeneration, and to enhance the efficiency of neural stem cell (NSC) transplantation. However, to acquire sufficient number of seed cells on the material appears to be the main obstacle to constructing functional transplantable NSC-biomaterial complexes. A culture system has been optimized in the current study to maintain the specific characteristics of NSCs/neural progenitor cells (NPCs) on the material and achieve sustaining increased multipotent seed cells. METHODS: The PHBHHx film was selected as biomaterial and the surface was firstly modified with NaOH treatment. The NSCs/NPCs isolated from the cerebral cortex of rat embryos were cultured on the treated PHBHHx films in growth medium containing 1%, 5%, and 10% fetal bovine serum (FBS). Then the attachment, survival, proliferation, and differentiation of NSCs/NPCs were assessed. RESULTS: NaOH treatment significantly increased the hydrophilicity of PHBHHx and enhanced NSCs/NPCs attachment. On the treated PHBHHx film, NSCs/NPCs survived well and actively proliferated in the medium containing 1% FBS. After 7-14 days in culture, approximately two-thirds of cells remained as nestin and Sox2 positive NSCs/NPCs. However, in the medium containing 5% and 10% FBS, NSCs/NPCs proliferation was reduced and differentiation, particularly glial differentiation was significantly promoted. CONCLUSION: Growth medium containing low concentration of FBS is favorable for maintaining the characteristics, in terms of self-renewal and multiple differentiation, of NSCs/NPCs on NaOH-treated PHBHHx films. This could be a useful method to construct functional transplantable NSCs/NPCs-biomaterial complex.