Research on aggregation-induced emission (AIE) has become increasingly popular recently and various AIE luminogens (AIEgens) have been developed based on tetraphenylethene, hexaphenylsilole, distyrylanthracene, tetraphenylpyrazine, etc. However, facile tuning of the AIEgen emissions in a wide range remains challenging. Herein, a novel series of AIEgens is reported, based on imidazole-cored molecular rotors, with facile synthesis and emission colors covering the whole visible spectrum. Moreover, these imidazole derivatives exhibit biological functions unique among the AIEgens, including mitochondria-specific imaging and antifungal activity. Benefiting from the easy preparation and the tunable emission, the imidazole derivatives are expected to not only diversify the family of AIEgens but also enrich their biological applications.

Bioorthogonal turn-on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn-on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn-on probes based on red-emissive fluorogens with aggregation-induced emission characteristics (AIEgens). The probe is water soluble and non-fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide-functionalized glycans on cancer cell surface. The fluorescence turn-on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400–700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.

Bioorthogonal turn-on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn-on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn-on probes based on red-emissive fluorogens with aggregation-induced emission characteristics (AIEgens). The probe is water soluble and non-fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide-functionalized glycans on cancer cell surface. The fluorescence turn-on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400–700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.

Multimodal imaging provides complimentary information that is advantageous in studying both cellular and molecular mechanisms in vivo, which has tremendous potential in pre-clinical research and clinical translational imaging. It is desirable to design probes for multimodal imaging that can be administered minimally but provides multifaceted information. Herein, we demonstrate the complementary dual functional ability of a nanoconstruct for molecular imaging in both photoacoustic (PA) and surface-enhanced Raman scattering (SERS) biosensing simultaneously in tandem. To realize this, a group of NIR active organic molecules are designed and synthesized that possess both SERS and PA activity. Nanoconstructs realized by anchoring such molecules onto gold nanoparticles are demonstrated for targeting cancer biomarkers in vivo while providing complimentary information about biodistribution and targeting efficiency. In future, such nanoconstructs could play a major role in identifying surgical margins and also for disease monitoring in translational medicine.

Compared with traditional one-photon fluorescence imaging, two-photon fluorescence imaging techniques have shown advantages such as increased penetration depth, lower tissue autofluorescence, and reduced photo­damage, and therefore are particularly useful for imaging tissues and animals. In this work, the design and synthesis of two novel DPP-based compounds with large two-photon absorption (2PA) cross-sections (σ ≥ 8100 GM) and aggregation-induced emission (AIE) properties are reported. The new compounds are red/NIR emissive and show large Stokes shifts (Δλ ≥ 3571 cm⁻¹). 1,2-Distearoyl-sn-glycero-3-phosphoethanol amine-N-[maleimide(polyethylene glycol)-2000 (DSPE-PEG-Mal) is used as the encapsulation matrix to encapsulate DPP-2, followed by surface functionalization with cell penetrating peptide (CPP) to yield DPP-2-CPP nanoparticles with high brightness, good water dispersibility, and excellent biocompatibility. DPP-2 nanoparticles have been used for cell imaging and two-photon imaging with clear visualization of blood vasculature inside mouse ear skin with a depth up to 80 μm.

Stem cell–based therapies hold great promise in providing desirable solutions for diseases that cannot be effectively cured by conventional therapies. To maximize the therapeutic potentials, advanced cell tracking probes are essential to understand the fate of transplanted stem cells without impairing their properties. Herein, conjugated polymer (CP) nanodots are introduced as noninvasive fluorescent trackers with high brightness and low cytotoxicity for tracking of mesenchymal stem cells (MSCs) to reveal their in vivo behaviors. As compared to the most widely used commercial quantum dot tracker, CP nanodots show significantly better long-term tracking ability without compromising the features of MSCs in terms of proliferation, migration, differentiation, and secretome. Fluorescence imaging of tissue sections from full-thickness skin wound–bearing mice transplanted with CP nanodot-labeled MSCs suggests that paracrine signaling of the MSCs residing in the regenerated dermis is the predominant contribution to promote skin regeneration, accompanied with a small fraction of endothelial differentiation. The promising results indicate that CP nanodots could be used as next generation of fluorescent trackers to reveal the currently ambiguous mechanisms in stem cell therapies through a facile and effective approach.

Integrated systems that offer traceable cancer therapy are highly desirable for personalized medicine. Herein, a probe is reported that is composed of a red-emissive photosensitizer (PS) with aggregation-induced emission characteristics and a built-in apoptosis sensor with activatable green emission for targeted cancer cell ablation and real-time monitoring of PS activation and therapeutic response. The probe is nonemissive in aqueous media and can be selectively uptaken by α_vβ₃ integrin overexpressed cancer cells. Cleavage of the probe by intracellular glutathione leads to release of the apoptosis sensor and red fluorescence turn-on to report the PS activation. Upon light irradiation, the PS can generate reactive oxygen species to induce cell apoptosis and activate caspase-3/-7, which will cleave the apoptosis sensor to yield intense green fluorescence. Both the red and green emission can be obtained through a single wavelength excitation, which makes the probe very convenient for therapeutic protocol development.

The design and synthesis is reported for a fluorescent conjugated polymer (CP), poly{[4,4,9,9-tetrakis(4-(octyloxy)phenyl-4,9-dihydro-s-indaceno[1,2-b:5,6-b′]dithiophene)]-alt-co-[4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole]} (PIDT-DBT), with absorption and emission profiles fallen within far-red/near infrared (FR/NIR) region and further demonstrate its application in long-term in vitro cell tracing and in vivo imaging of liver tumor growth. PIDT-DBT-Tat nanoparticles (NPs) have an absorption maximum at ≈600 nm with an emission maximum at ≈720 nm in water. In vitro cell tracing studies reveal that PIDT-DBT-Tat NPs can trace HepG2 liver cancer cells over 8 d. In vivo imaging results indicate that PIDT-DBT-Tat NPs can monitor liver tumor growth for more than 27 d in a real-time manner. Both in vitro and in vivo studies demonstrate that PIDT-DBT-Tat NPs are superior to commercial Qtracker 705 as fluorescent probes. This study demonstrates for the first time the feasibility for long-term in vivo imaging of tumor growth by utilizing CP-based fluorescent probes, which will encourage the development of NIR fluorescent CPs for in vivo bioimaging.

Targeted delivery of drugs toward mitochondria of specific cancer cells dramatically improves therapy efficiencies especially for photodynamic therapy (PDT), as reactive oxygen species (ROS) are short in lifetime and small in radius of action. Different from chemical modification, nanotechnology has been serving as a simple and nonchemical approach to deliver drugs to cells of interest or specific organelles, such as mitochondria, but there have been limited examples of dual-targeted delivery for both cells and mitochondria. Here, cellular and mitochondrial dual-targeted organic dots for image-guided PDT are reported based on a fluorogen with aggregation-induced emission (AIEgen) characteristics. The AIEgen possesses enhanced red fluorescence and efficient ROS production in aggregated states. The AIE dot surfaces are functionalized with folate and triphenylphosphine, which can selectively internalize into folate-receptor (FR) positive cancer cells, and subsequently accumulate at mitochondria. The direct ROS generation at mitochondria sites is found to depolarize mitochondrial membrane, affect cell migration, and lead to cell apoptosis and death with enhanced PDT effects as compared to ROS generated randomly in cytoplasm. This report demonstrates a simple and general nanocarrier approach for cellular and mitochondrial dual-targeted PDT, which opens new opportunities for dual-targeted delivery and therapy.

Endo/lysosomal escape of gene vectors and the subsequent unpacking of nucleic acids in cytosol are two major challenges for efficient gene delivery. Herein, we report a polymeric gene delivery vector, which consists of a photosensitizer (PS) with aggregation-induced emission (AIE) characteristics and oligoethylenimine (OEI) conjugated via an aminoacrylate (AA) linker that can be cleaved by reactive oxygen species (ROS). In aqueous media, the polymer could self-assemble into bright red fluorescent nanoparticles (NPs), which can efficiently bind to DNA through electrostatic interaction for gene delivery. Upon visible light irradiation, the generated ROS can break the endo/lysosomal membrane and the polymer, resulting in light-controlled endo/lysosomal escape and unpacking of DNA for efficient gene delivery. The smart polymer represents the first successful gene vector to simultaneously address both challenges with a single light excitation process.

Endo/lysosomal escape of gene vectors and the subsequent unpacking of nucleic acids in cytosol are two major challenges for efficient gene delivery. Herein, we report a polymeric gene delivery vector, which consists of a photosensitizer (PS) with aggregation-induced emission (AIE) characteristics and oligoethylenimine (OEI) conjugated via an aminoacrylate (AA) linker that can be cleaved by reactive oxygen species (ROS). In aqueous media, the polymer could self-assemble into bright red fluorescent nanoparticles (NPs), which can efficiently bind to DNA through electrostatic interaction for gene delivery. Upon visible light irradiation, the generated ROS can break the endo/lysosomal membrane and the polymer, resulting in light-controlled endo/lysosomal escape and unpacking of DNA for efficient gene delivery. The smart polymer represents the first successful gene vector to simultaneously address both challenges with a single light excitation process.

Activatable photosensitizers (PSs) have been widely used for the simultaneous fluorescence imaging and photodynamic ablation of cancer cells. However, the ready aggregation of traditional PSs in aqueous media can lead to fluorescence quenching as well as reduced phototoxicity even in the activated form. We have developed a series of PSs that show aggregation-enhanced emission and phototoxicity and thus the exact opposite behavior to that of previously reported PSs. We further developed a dual-targeted enzyme-activatable bioprobe based on the optimized photosensitizer and describe simultaneous light-up fluorescence imaging and activated photodynamic therapy for specific cancer cells. The design of smart probes should thus open new opportunities for targeted and image-guided photodynamic therapy.

Activatable photosensitizers (PSs) have been widely used for the simultaneous fluorescence imaging and photodynamic ablation of cancer cells. However, the ready aggregation of traditional PSs in aqueous media can lead to fluorescence quenching as well as reduced phototoxicity even in the activated form. We have developed a series of PSs that show aggregation-enhanced emission and phototoxicity and thus the exact opposite behavior to that of previously reported PSs. We further developed a dual-targeted enzyme-activatable bioprobe based on the optimized photosensitizer and describe simultaneous light-up fluorescence imaging and activated photodynamic therapy for specific cancer cells. The design of smart probes should thus open new opportunities for targeted and image-guided photodynamic therapy.

Efficient long-term cell tracing in a noninvasive and real-time manner is of great importance to understand genesis, development, invasion, and metastasis of cancerous cells. Cell penetrating organic dots with aggregation- induced emission (AIE) characteristics are successfully developed as long-term cell trackers. The AIE dots enjoy the advantages of high emission efficiency, large Stokes shift, good biocompatibility, and high photostability, which ensure their good performance in long-term non-invasive in vitro cell tracing. Moreover, it is the first report that AIE dots exhibit certain permeability to cellular nucleus, making them attractive potential candidates for nucleus imaging. The AIE dots display superior performance compared to their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for monitoring biological processes.

Nanomaterials that combine diagnostic and therapeutic functions within a single nanoplatform are highly desirable for molecular medicine. Herein we report a novel theranostic platform based on a conjugated-polyelectrolyte (CPE) polyprodrug that contains functionality for image, chemo- and photodynamic therapy (PDT), and on-demand drug release upon irradiation with a single light source. Specifically, the PEGylated CPE serves as a photosensitizer and a carrier, and is covalently conjugated to doxorubicin through a linker that can be cleaved by reactive oxygen species (ROS). Under appropriate light irradiation, the CPE can generate ROS, not only for PDT, but also for on-demand drug release and chemotherapy. This nanoplatform will offer on-demand PDT and chemotherapy with drug release triggered by one light switch, which has great potential in cancer treatment.

Subcellular organelle-specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria-targeting probe (AIE-mito-TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation-induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE-mito-TPP probe can selectively accumulate in cancer-cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE-mito-TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light-up probe provides a unique strategy for potential image-guided therapy of cancer cells.

Nanomaterials that combine diagnostic and therapeutic functions within a single nanoplatform are highly desirable for molecular medicine. Herein we report a novel theranostic platform based on a conjugated-polyelectrolyte (CPE) polyprodrug that contains functionality for image, chemo- and photodynamic therapy (PDT), and on-demand drug release upon irradiation with a single light source. Specifically, the PEGylated CPE serves as a photosensitizer and a carrier, and is covalently conjugated to doxorubicin through a linker that can be cleaved by reactive oxygen species (ROS). Under appropriate light irradiation, the CPE can generate ROS, not only for PDT, but also for on-demand drug release and chemotherapy. This nanoplatform will offer on-demand PDT and chemotherapy with drug release triggered by one light switch, which has great potential in cancer treatment.

Subcellular organelle-specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria-targeting probe (AIE-mito-TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation-induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE-mito-TPP probe can selectively accumulate in cancer-cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE-mito-TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light-up probe provides a unique strategy for potential image-guided therapy of cancer cells.

The application of a time-resolved photoluminescence technique and fluorescence lifetime imaging microscopy for biosensing and bioimaging based on phosphorescent conjugated polyelectrolytes (PCPEs) containing Ir(III) complexes and polyfluorene units is reported. The specially designed PCPEs form 50 nm nanoparticles with blue fluorescence in aqueous solutions. Electrostatic interaction between the nanoparticles and heparin improves the energy transfer between the polyfluorene units to Ir(III) complex, which lights up the red signal for naked-eye sensing. Good selectivity has been demonstrated for heparin sensing in aqueous solution and serum with quantification ranges of 0–70 μM and 0–5 μM, respectively. The signal-to-noise ratio can be further improved through time-resolved emission spectra, especially when the detection is conducted in complicated environment, e.g., in the presence of fluorescent dyes. In addition to heparin sensing, the PCPEs have also been used for specific labeling of live KB cell membrane with high contrast using both confocal fluorescent cellular imaging and fluorescence lifetime imaging microscopies. This study provides a new perspective for designing promising CPEs for biosensing and bioimaging applications.

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.

Understanding the localization and engraftment of tumor cells at postintravasation stage of metastasis is of high importance in cancer diagnosis and treatment. Advanced fluorescent probes and facile methodologies for cell tracing play a key role in metastasis studies. In this work, we design and synthesize a dual-modality imaging dots with both optical and magnetic contrast through integration of a magnetic resonance imaging reagent, gadolinium(III), into a novel long-term cell tracing probe with aggregation-induced emission (AIE) in far-red/near-infrared region. The obtained fluorescent-magnetic AIE dots have both high fluorescence quantum yield (25%) and T₁ relaxivity (7.91 mM⁻¹ s⁻¹) in aqueous suspension. After further conjugation with a cell membrane penetrating peptide, the dual-modality dots can be efficiently internalized into living cells. The gadolinium(III) allows accurate quantification of biodistribution of cancer cells via intraveneous injection, while the high fluorescence provides engraftment information of cells at single cellular level. The dual-modality AIE dots show obvious synergistic advantages over either single imaging modality and hold great promises in advanced biomedical studies.

Light emission of 2-(2,6-bis((E)-4-(diphenylamino)styryl)-4H-pyran-4-ylidene)malononitrile (TPA-DCM) is weakened by aggregate formation. Attaching tetraphenylethene (TPE) units as terminals to TPA-DCM dramatically changes its emission behavior: the resulting fluorogen, 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), is more emissive in the aggregate state, showing the novel phenomenon of aggregation-induced emission (AIE). Formulation of TPE-TPA-DCM using bovine serum albumin (BSA) as the polymer matrix yields uniformly sized protein nanoparticles (NPs) with high brightness and low cytotoxicity. Applications of the fluorogen-loaded BSA NPs for in vitro and in vivo far-red/near-infrared (FR/NIR) bioimaging are successfully demonstrated using MCF-7 breast-cancer cells and a murine hepatoma-22 (H₂₂)-tumor-bearing mouse model, respectively. The AIE-active fluorogen-loaded BSA NPs show an excellent cancer cell uptake and a prominent tumor-targeting ability in vivo due to the enhanced permeability and retention effect.

A facile strategy is developed to synthesize dual-modal fluorescent-magnetic nanoparticles (NPs) with surface folic acid by co-encapsulation of a far-red/near-infrared (FR/NIR)-emissive conjugated polymer (PFVBT) and lipid-coated iron oxides (IOs) into a mixture of poly(lactic-co-glycolic-acid)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) and PLGA. The obtained NPs exhibit superparamagnetic properties and high fluorescence, which indicates that the lipid coated on IOs is effective at separating the conjugated polymer from IOs to minimize fluorescence quenching. These NPs are spherical in shape with an average diameter of ≈180 nm in water, as determined by laser light scattering. In vitro studies reveal that these dual-modal NPs can serve as an effective fluorescent probe to achieve targeted imaging of MCF-7 breast cancer cells without obvious cytotoxicity. In vivo fluorescence and magnetic resonance imaging results suggest that the NPs are able to preferentially accumulate in tumor tissues to allow dual-modal detection of tumors in a living body. This demonstrates the potential of conjugated polymer based dual-modal nanoprobes for versatile in vitro and in vivo applications in future.

A glucopyranose functionalized star-shaped oligomer, N-tris{4,4′,4′′-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-2-amido-2-deoxy-1-thio-β-D-glucopyranose-hexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylamine (TVFVBN-S-NH₂), is synthesized for two-photon fluorescence imaging. In water, TVFVBN-S-NH₂ self-assembles into nanoparticles with an average diameter of ∼49 nm and shows a fluorescence quantum yield of 0.21. Two-photon fluorescence measurements reveal that TVFVBN-S-NH₂ has a two-photon absorption cross-section of ∼1100 GM at 780 nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN-S-NH₂ with folic acid to yield TVFVBN-S-NH₂FA with similar optical and physical properties as those for TVFVBN-S-NH₂. Cellular imaging studies reveal that TVFVBN-S-NH₂FA has increased uptake by MCF-7 cells relative to that for TVFVBN-S-NH₂, due to specific interactions between folic acid and folate receptors on the MCF-7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water-soluble materials with a large two-photon absorption (TPA) cross-section for targeted cancer-cell imaging.

The synthesis of polyhedral oligomeric silsesquioxanes (POSS)-containing conjugated polymer (CP) and the polymer loaded poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) with surface antibody functionalization for human epidermal growth factor receptor 2 (HER2)-positive cancer cell detection are reported. Due to the steric hindrance of POSS, NPs prepared from POSS-containing CP show improved photoluminescence quantum yield as compared to that for the corresponding linear CP encapsulated NPs. In addition, the amount of -NH₂ groups on NP surface is well-controlled by changing the molar ratio of poly(lactic-co-glycolic-acid)-b-poly(ethylene glycol) (PLGA-b-PEG-NH₂) to PLGA-OCH₃ during NP formulation. Further conjugation of the NH₂-functionalized CP NPs with trastuzumab (Herceptin) yields NPs with fine-tuned protein density. These NPs are able to discriminate SKBR-3 breast cancer cells from MCF-7 breast cancer cells and NIH/3T3 fibroblast cells both on substrate and in suspension by taking advantage of the specific binding affinity between trastuzumab and HER2 overexpressed in SKBR-3 breast cancer cell membrane. The high quantum yield and fine-tuned surface specific protein functionalization make the POSS-containing CP loaded NPs a good candidate for targeted biological imaging and detection.

The synthesis of polyhedral oligomeric silsesquioxanes (POSS)-containing conjugated polymer (CP) and the polymer loaded poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) with surface antibody functionalization for human epidermal growth factor receptor 2 (HER2)-positive cancer cell detection are reported. Due to the steric hindrance of POSS, NPs prepared from POSS-containing CP show improved photoluminescence quantum yield as compared to that for the corresponding linear CP encapsulated NPs. In addition, the amount of -NH₂ groups on NP surface is well-controlled by changing the molar ratio of poly(lactic-co-glycolic-acid)-b-poly(ethylene glycol) (PLGA-b-PEG-NH₂) to PLGA-OCH₃ during NP formulation. Further conjugation of the NH₂-functionalized CP NPs with trastuzumab (Herceptin) yields NPs with fine-tuned protein density. These NPs are able to discriminate SKBR-3 breast cancer cells from MCF-7 breast cancer cells and NIH/3T3 fibroblast cells both on substrate and in suspension by taking advantage of the specific binding affinity between trastuzumab and HER2 overexpressed in SKBR-3 breast cancer cell membrane. The high quantum yield and fine-tuned surface specific protein functionalization make the POSS-containing CP loaded NPs a good candidate for targeted biological imaging and detection.

This Feature Article summarizes the recent advances of water-soluble fluorescent conjugated polyelectrolytes (CPEs) in bioimaging. Apart from a brief overview of traditional linear CPEs, a special emphasis is placed on CPEs that can self-assemble into or are born with three-dimensional nano-architectures, including grafted CPEs, hyperbranched CPEs, and polyhedral oligomeric silsesquioxanes(POSS)-based CPE derivatives. These CPEs naturally form nanoparticles with sizes ranging from 3 to 100 nm in aqueous media, and possess reactive functional groups for bioconjugation or complexation with desired biorecognition elements. The tunable size, low cytotoxicity, good photostability, and ease of surface modification ultimately enable these CPEs with wide applications in in vitro intracellular protein sensing, cell detection, in vivo cell imaging and drug tracking. Moreover, traditional linear CPEs can be transformed into uniform nanoparticles by complexation with oppositely charged biomolecules to allow for cell detection and in situ drug release monitoring. The work featured herein not only reveals the important molecular design principles of CPEs for different imaging tasks, but also highlights the promising directions for the further development of CPE-based imaging materials.

A highly sensitive strand specific DNA assay, which consists of a peptide nucleic acid (PNA) probe, a cationic conjugated polymer (PFVP), and self-assembled polystyrene beads in microwell arrays on silicon chip, is reported. PFVP, as an efficient signal amplifier and signal reporter, has been specially designed and synthesized to be compatible with commercial confocal microscopes for sensing on solid substrates. The assay operates on the net increase in negative charge at the PNA surface that occurs upon single-stranded DNA hybridization, which subsequently allows complex formation with the positively charged PFVP to favor energy transfer between the polymer and Cy5-labeled target. With maximized surface contact provided by bead arrays and signal amplification provided by PFVP, this assay allows detection of ∼300 copies of Cy5-labeled DNA using a commercial confocal microscope. In addition, the same strategy is also extended for label-free DNA detection with a detection sensitivity of 150 attomole. Excellent discrimination against single nucleotide polymorphism (SNP) is also demonstrated for both Cy5-labeled and label-free target detection. This study indicates that cationic conjugated polymers have great potential to be incorporated into the widely used microarray technology for simplified process with improved detection sensitivity.

A red-fluorescent conjugated polyelectrolyte (CPE, P2) is grafted with dense poly(ethylene glycol) (PEG) chains via click chemistry and subsequently modified with folic acid to form a molecular brush based cellular probe (P4). P4 self-assembles into a core–shell nanostructure in aqueous medium with an average size of 130 nm measured by laser light scattering. As compared to P2, P4 possesses not only a substantially higher quantum yield (11%), but also reduced nonspecific interactions with biomolecules in aqueous medium due to the shielding effect of PEG. In conjunction with its high photostability and low cytotoxicity, utilization of P4 as a far-red/near-infrared cellular probe allows for effective visualization and discrimination of MCF-7 cancer cells from NIH-3T3 normal cells in a high contrast, selective, and nonviral manner. This study thus demonstrates a flexible molecular brush approach to overcome the intrinsic drawbacks of CPEs for advanced bioimaging applications.

A red-fluorescent conjugated polyelectrolyte (CPE, P2) is grafted with dense poly(ethylene glycol) (PEG) chains via click chemistry and subsequently modified with folic acid to form a molecular brush based cellular probe (P4). P4 self-assembles into a core–shell nanostructure in aqueous medium with an average size of 130 nm measured by laser light scattering. As compared to P2, P4 possesses not only a substantially higher quantum yield (11%), but also reduced nonspecific interactions with biomolecules in aqueous medium due to the shielding effect of PEG. In conjunction with its high photostability and low cytotoxicity, utilization of P4 as a far-red/near-infrared cellular probe allows for effective visualization and discrimination of MCF-7 cancer cells from NIH-3T3 normal cells in a high contrast, selective, and nonviral manner. This study thus demonstrates a flexible molecular brush approach to overcome the intrinsic drawbacks of CPEs for advanced bioimaging applications.

A neutral polyfluorene derivative that contains 20 mol % 2,1,3-benzothiadiazole (BT) is synthesized by Suzuki cross-coupling polymerization. A cationic conjugated polymer A and an α-mannose-bearing polymer B are subsequently obtained through different post-polymerization methods. As a result of the charged pendant groups or sugar-bearing groups attached to the polymer side chains, both A and B show good water-solubility. The titration of Concanavalin A (Con A) into polymer aqueous solution leads to different fluorescent responses for polymers A and B. Polymer A does not show any obvious fluorescence change upon interaction with Con A, whereas polymer B shows fluorescence increase in BT emission intensity when Con A is added. This is because of the specific interaction between α-mannose and Con A, which induces polymer aggregation, and then facilitates energy transfer from the phenylene–fluorene segments to the BT units. A practical calibration curve ranging from 1 nM to 250 nM is obtained by correlating the changes in BT emission intensity with Con A concentration. The advantage of polymer B-based Con A macromolecular probe is that it shows signal increase upon Con A recognition, which is significantly different from other conjugated polymer-based fluorescence quenching assays.

Thiazole orange (TO), an intercalating dye, is integrated into cationic poly(fluorene-alt-phenylene) (PFP) to develop a macromolecular multicolor probe (PFPTO) for double-stranded DNA (dsDNA) detection. This polymer design not only takes advantage of the high affinity between TO and dsDNA to realize dsDNA recognition in biological media, but also brings into play the light-harvesting feature of conjugated polymers to amplify the signal output of TO in situ. PFPTO differentiates dsDNA from single-stranded DNA (ssDNA) more effectively upon excitation of the conjugated backbone relative to that upon direct excitation of TO as a result of efficient fluorescence resonance energy transfer from the polymer backbone to the intercalated TO. In the presence of dsDNA, energy transfer within PFPTO is more efficient as compared to that for free TO/PFP system, which leads to better dsDNA discriminability for PFPTO in contrast to that for TO/PFP. The distinguishable fluorescent color for PFPTO solutions in the presence of dsDNA allows naked-eye detection of dsDNA with the assistance of a hand-held UV lamp. The significant advantage of this macromolecular fluorescent probe is that naked-eye detection of label-free dsDNA can be performed in biological media in real-time.

Two cationic poly(fluorene-alt-benzothiadiazole)s with different side chains are designed and synthesized. Both polymers show low fluorescence in aqueous solution due to the charge-transfer character of the polymer's excited states. Fluorescence turn-on biosensors for heparin detection and quantification are developed, taking advantage of complexation-induced aggregation, which increases the polymer fluorescence in aqueous solution. It is found that good polymer water-solubility is beneficial to the sensitivity and fluorescence contrast of the heparin turn-on sensor as a result of the low fluorescence background. Moreover, stronger complexation between the polymer/heparin leads to a substantially larger fluorescence increase in the presence of heparin relative to that in the presence of its analog, hyaluronic acid (HA), allowing discrimination of heparin from HA. Heparin quantification with a practical calibration range covering the whole therapeutic dosing levels (0.2–8 U mL⁻¹) is realized based on the polymer with good water-solubility. This investigation provides a new insight for designing conjugated polymers with a light-up signature for biomolecular sensing.

A general strategy for the preparation of highly fluorescent poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (NPs) loaded with conjugated polymers (CPs) is reported. The process involves encapsulation of organic-soluble CPs with PLGA using a modified solvent extraction/evaporation technique. The obtained NPs are stable in aqueous media with biocompatible and functionalizable surfaces. In addition, fluorescent properties of the CP-loaded PLGA NPs (CPL NPs) could be fine-tuned by loading different types of CPs into the PLGA matrix. Four types of CPL NPs are prepared with a volume-average hydrodynamic diameter ranging from 243 to 272 nm. The application of CPL NPs for bio-imaging is demonstrated through incubation with MCF-7 breast cancer cells. Confocal laser scanning microscopy studies reveal that the CPL NPs are internalized in cytoplasm around the nuclei with intense fluorescence. After conjugation with folic acid, cellular uptake of the surface-functionalized CPL NPs is greatly enhanced via receptor-mediated endocytosis by MCF-7 breast cancer cells, as compared to that for NIH/3T3 fibroblast cells, which indicates a selective targeting effect of the folate-functionalized CPL NPs in cellular imaging. The merits of CPL NPs, such as low cytotoxicity, high fluorescence, good photostability, and feasible surface functionalization, will inspire extensive study of CPL NPs as a new generation of probes for specific biological imaging and detection.

We report a convenient and effective method to enhance the signal output of dye-labeled oligonucleotide sensitized by cationic conjugated polymers (CCP). Sodium dodecyl sulphate (SDS) is utilized to regulate the interaction between CCP and dye-labeled single-stranded DNA in order to reduce the dye self-quenching within the CCP/DNA complexes. Improvement of CCP-sensitized dye emissison in the presence of SDS relative to that in the absence of SDS is observed, which reveals the importance of reducing CCP charge density in improving the energy transfer from CCP to dye-labeled probes.

Cationic conjugated polymers (CCPs) with different charge densities are synthesized via Suzuki polymerization. The CCPs show similar optical properties in aqueous solutions but obvious difference in fluorescence resonance energy transfer (FRET) to Texas Red-labeled single-stranded DNA (ssDNA-TR). Both CCP and TR fluorescence quenching are revealed to influence the energy-transfer process. The difference in quantum yields of CCP/ssDNA complexes highlights the importance of polymer side-chain structures and charge density. A CCP with a high charge density and ethylene oxide as the side chain provides the highest quantum yield for CCP/ssDNA complexes, which favors FRET. TR quenching within the CCP/ssDNA complexes is predominantly determined by the CCP charge density. In contrast to the other two polymers, the CCP with low charge density provides the most-intense polymer-sensitized TR emission, which is due to the collective response of more optically active polymer units around TR and the minimized TR self-quenching within the CCP/ssDNA-TR complexes. These studies provide a new guideline for improving the signal amplification of conjugated-polymer-based optical sensors.

We have developed a new intermediate monomer, 2,7-[bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis(3-(tert-butyl propanoate))]fluorene, that allows the easy synthesis of water-soluble carboxylated polyfluorenes. As an example, poly[9,9′-bis(3′′-propanoate)fluoren-2,7-yl] sodium salt was synthesized by the Suzuki coupling reaction, and the properties of the polymer were studied in aqueous solutions of different pH. Fluorescence quenching of the polymer by different cationic quenchers (MV²⁺, MV⁴⁺, and NO₂MV²⁺; MV=methyl viologen) was studied, and the quenching constants were found to be dependent on the charge and electron affinity of the quencher molecule and the pH of the medium. The largest quenching constant was observed to be 1.39×10⁸ M⁻¹ for NO₂MV²⁺ at pH 7. The change in polymer fluorescence upon interaction with different proteins was also studied. Strong fluorescence quenching of the polymer was observed in the presence of cytochrome c, whereas weak quenching was observed in the presence of myoglobin and bovine serum albumin. Lysozyme quenched the polymer emission at low protein concentrations, and the quenching became saturated at high protein concentrations. Under similar experimental conditions, the polymer showed improved quenching efficiencies toward cationic quenchers and a more selective response to proteins relative to other carboxylated conjugated polymers.

A simple and efficient approach was developed for the synthesis of a series of cationic water-soluble oligofluorenes up to a chain length of a heptamer. Bromoalkyl-substituted fluorenyl boronic esters as the key intermediates were synthesized by using a modified Miyaura reaction. With an increasing number of repeat units (trimer to hexamer), the size-specific oligomers have shown redshifts in both the absorption and emission maxima. The emission maximum reaches the limit for the hexamer in both water and buffer solution. The quantum yields of the oligomers decreased with increased oligomer size in water. Both fluorescence quenching of the oligomers by 9,10-anthraquinone-2,6-disulfonate and the fluorescence resonance energy transfer experiments with the oligomers as the donor and fluorescein (Fl)-labeled double-stranded DNA (dsDNA-Fl) as the acceptor revealed the chain-length-dependent behavior. The Stern–Volmer quenching constant increased with the molecular size, whereas the highest donor-sensitized Fl emission was observed for the hexamer. These size-specific oligomers also served as a model to study the structure–property relationships for cationic polyfluorenes.