A light-up endoplasmic reticulum probe based on a rational design of red-emissive fluorogens with aggregation-induced emission.

Fine-tuning electron acceptors through changing one cyano group to an amide generates a more stable and emissive fluorophore with the character of aggregation-induced emission. Conjugation between the new fluorophore and CFFKDEL generated an excellent ER targeting light-up probe with high specificity and good photostability.

End Functionalized Nonionic Water-Dispersible Conjugated Polymers.

2,7-Dibromofluorene monomers carrying two or four oligo(ethylene glycol) (OEG) side chains are synthesized. Heck coupling between the monomers and 1,4-divinylbenzene followed by end capping with [4-(4-bromophenoxy)butyl]carbamic acid tert-butyl ester leads to two nonionic water-dispersible poly(fluorene-alt-1,4-divinylenephenylene)s end-functionalized with amine groups after hydrolysis. In water, the polymer with a lower OEG density (P1) has poor water dispersibility with a quantum yield of 0.24, while the polymer with a higher OEG density (P2) possesses excellent water-dispersibility with a high quantum yield of 0.45. Both polymers show fluorescence enhancement and blue-shifted absorption and emission maxima in the presence of surfactant sodium dodecyl sulfate and dodecyltrimethylammonium bromide. The polymers are also resistant to ionic strength with minimal nonspecific interactions to bovine serum albumin. When biotin is incorporated into the end of the polymer backbones through N-hydroxysuccinimide/amine coupling reaction, the biotinylated polymers interact specifically with streptavidin on solid surface.

AIE Polymers: Synthesis, Properties, and Biological Applications.

Aggregation-caused quenching (ACQ) is a general phenomenon that is faced by traditional fluorescent polymers. Aggregation-induced emission (AIE) is exactly opposite to ACQ. AIE molecules are almost nonemissive in their molecularly dissolved state, but they can be induced to show high fluorescence in the aggregated or solid state. Incorporation of AIE phenomenon into polymer design has yielded various polymers with AIE characteristics. In this review, the recent progress of AIE polymers for biological applications is summarized.

Functionalized Conjugated Polyelectrolytes for Biological Sensing and Imaging.

Conjugated polyelectrolytes (CPEs) are macromolecules with highly delocalized π-conjugated backbones and charged side chains, which are unique types of active materials, with wide applications in optoelectronics, sensing, imaging, and therapy. By attaching specific groups (e.g., recognition elements, magnetic resonance (MR) contrast agents, gene carriers, and drugs) to the side chain or backbone of CPEs, functionalized CPEs have been developed and used for specific biological applications. In this account, we summarize the recent progress of functionalized CPEs with respect to their synthesis and biomedical applications. Future perspectives are also discussed at the end.

Narrow band gap conjugated polyelectrolytes for photothermal killing of bacteria.

We report the demonstration of antimicrobial conjugated polyelectrolytes (CPEs) with high NIR absorbance for selective and efficient photothermal killing of bacteria over mammalian cells. The antimicrobial CPE possessing quaternary ammonium (QA) terminated side chains (P1) shows higher binding preference and increased dark toxicity towards Gram-positive and Gram-negative bacteria over mammalian cells. Bestowed by π-conjugated backbones, P1 exhibits a high molar absorptivity of 39.8 L g-1 cm-1 at 808 nm with an efficient photothermal conversion efficiency of 33 ± 1%. Upon 808 nm laser irradiation, P1 shows enhanced bactericidal effects, but not to mammalian cells. Although the anionic CPE counterpart with the same polymer backbone but sulfonate terminated side chains (P2) possesses a similar photothermal conversion ability, it exhibits much lower antibacterial effects due to its low binding affinity. This study thus reveals that bacteria-CPE electrostatic interactions play a major role in bacterial recognition, although hydrophobic interactions also contribute.

Water-soluble bioprobes with aggregation-induced emission characteristics for light-up sensing of heparin.

Two water-soluble cationic fluorene-based fluorescent probes for heparin detection are designed and synthesized. A slight change in the molecular design results in two probes with opposite optical properties in their solution and aggregation states as well as a response to heparin in buffer solution. The probe with a propeller-like conformation exhibits aggregation-induced emission (AIE) characteristics and shows a green fluorescence enhancement upon interaction with heparin; in contrast, the probe with a more planar conformation has a fluorescence quenching response. A comprehensive study on heparin detection using the two probes was conducted, which revealed that the AIE probe shows a better performance than the aggregation-caused quenching (ACQ) probe in terms of sensitivity. The AIE probe integrated with graphene oxide (GO) further improves the heparin detection sensitivity and selectivity. The solution of AIE probe/GO emits strong green fluorescence only in the presence of heparin, which allows for light-up visual discrimination of heparin from its analogues such as chondroitin-4-sulfate and hyaluronic acid. Moreover, the linear light-up response of AIE probe/GO enables heparin quantification in the range of 0-13.2 µM with a detection limit of 10 nM, which is of practical importance for heparin monitoring during surgery or therapy.

Conjugated polymer amplified far-red/near-infrared fluorescence from nanoparticles with aggregation-induced emission characteristics for targeted in vivo imaging.

Fluorescence-amplified far-red/near-infrared (FR/NIR) nanoparticles (NPs) are synthesized by co-encapsulation of conjugated polymer donor (poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorenyldivinylene]; PFV) and a fluorogen acceptor (2-(2,6-bis((E)-4-(phenyl(4'-(1,2,2-triphenylvinyl)-[1,1'-biphenyl]-4-yl)amino)styryl)-4H-pyran-4-ylidene)malononitrile; TPE-TPA-DCM) with aggregation-induced emission (AIE) characteristics using biocompatible bovine serum albumin (BSA) as the encapsulation matrix. The good spectral overlap and close proximity between PFV and TPE-TPA-DCM in BSA NPs result in a 5.3-fold amplified TPE-TPA-DCM emission signal via fluorescence resonance energy transfer (FRET). The obtained PFV/TPE-TPA-DCM co-loaded BSA NPs are spherical in shape with a large Stokes shift of ∼223 nm and low cytotoxicity. The BSA matrix allows further functionalization with arginine-glycine-aspartic acid (RGD) peptide to yield fluorescent probes for specific recognition of integrin receptor-overexpressed cancer cells. The advantage of PFV amplified FR/NIR signal from TPE-TPA-DCM is further demonstrated in cellular and in vivo imaging using HT-29 colon cancer cells and a murine hepatoma H22 tumor-bearing mouse model, respectively. The high FR/NIR fluorescence and specific cancer targeting ability by RGD surface functionalization make the PFV/TPE-TPA-DCM co-loaded BSA-RGD NPs a unique FR/NIR fluorescent probe for cellular imaging and in vivo tumor diagnosis in a high contrast and selective manner.

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte.

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.

Butterfly-shaped conjugated oligoelectrolyte/graphene oxide integrated assay for light-up visual detection of heparin.

A water-soluble pyrene-based butterfly shaped conjugated oligoelectrolyte (TFP) is synthesized and integrated with graphene oxide (GO) to form a label-free assay for heparin detection. Efficient fluorescence quenching occurs between TFP and GO because of strong electrostatic and π-π interactions, leading to nearly dark emission in the absence of analytes. Addition of heparin into TFP solution significantly minimizes the fluorescence quenching of GO toward TFP, which is less effective for the heparin analogues, such as hyaluronic acid and chondroitin 4-sulfate. As a consequence, the solution emits strong yellow fluorescence only in the presence of heparin, which allows for light-up visual discrimination of heparin from its analogues. Moreover, the linear light-up response of the TFP/GO integrated assay enables heparin quantification in the range of 0-1.76 U/mL with a limit of detection of 0.046 U/mL, which is practical for heparin monitoring during postoperative and long-term care. This study thus demonstrates a new synthetic strategy to develop GO-based chemical and biological sensing without the employment of dye-labeled biomolecules.

Conjugated polymer loaded nanospheres with surface functionalization for simultaneous discrimination of different live cancer cells under single wavelength excitation.

Two conjugated polymers, poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl) fluorenyldivinylene] (PFV) and the PFV derivative containing 10 mol % 2,1,3-benzothiadiazole (BT) units (PFVBT), have been synthesized and employed to fabricate conjugated polymer loaded nanospheres for simultaneous discrimination of mixed live cancer cells in one solution. The incorporation of BT units into the PFV backbone leads to PFVBT with a similar absorption maximum but significantly red-shifted emission in film state as compared to those of PFV, due to aggregation enhanced energy transfer from the fluorenevinylene segments to electron-deficient BT units. Both conjugated polymer loaded nanospheres have shown optical features that are similar to their film states, which allow simultaneous multichannel signal collection with negligible interference upon excitation at a single wavelength. After further surface functionalization with antihuman epidermal growth factor receptor 2 (HER2) affibody or arginine-glycine-aspartic acid (RGD) peptide, the distinct fluorescence from PFV or PFVBT loaded nanospheres allows differentiation of SKBR-3 breast cancer cells (HER2 overexpression) from HT-29 colon cancer cells (integrin receptor overexpression) in live cell mixtures. The conjugated polymer loaded nanospheres with high quantum yield, low cytotoxicity, and multiple color emission upon single laser excitation are ideal for simultaneous multiple-target imaging and detection.

Conjugated polyelectrolyte blend as perturbable energy donor-acceptor assembly with multicolor fluorescence response to proteins.

Blending conjugated polyelectrolytes is demonstrated to be a convenient yet effective method to create a perturbable energy transfer systems with multicolor fluorescence response toward both nonmetalloproteins and metalloproteins, which holds great promise in visual protein sensing.

Homogeneous Detection of Trypsin in Protein Mixtures Based on Fluorescence Resonance Energy Transfer between Anionic Conjugated Polymer and Fluorescent Probe.

Herein we describe a novel and simple conjugated polymer-fluorescent probe based platform for trypsin detection from protein mixtures in homogeneous solution. This platform takes advantage of specific interaction between the probe and the active site of trypsin and the electrostatic interaction between the polymer and the protein to mediate energy transfer between the polymer and the probe. This method does not require any separation steps, which should facilitate high-throughput protease screening and drug discovery.

Naked-eye detection and quantification of heparin in serum with a cationic polythiophene.

A strategy for naked-eye detection and quantification of heparin in biological media, such as fetal bovine serum (FBS) is demonstrated by monitoring the absorbance change of a water-soluble cationic polythiophene. The negatively charged heparin interacts with positively charged polythiophene through electrostatic interaction, which leads to polymer conformation and color change from yellow to orange in solution. Under optimized conditions, addition of heparin derivatives, such as hyaluronic acid or chondroitin 4-sulfate to the same polymer solution leads to less change in polymer conformation and solution color due to their lower charge density as compared to that of heparin. Increasing the detection temperature or simply adding some organic solvent to the aqueous media reduces the polymer-polymer interchain pi stacking, and the polymer color change can be used to clearly differentiate heparin from its analogues in homogeneous solutions. Quantification of heparin is also demonstrated by correlating the changes in polymer absorbance to the heparin concentration. A linear calibration curve is observed in the 0-6.7 U/mL and 0-2.2 U/mL ranges for heparin quantification in pure water and in FBS, respectively.