A bright chemiluminescence conjugated polymer-mesoporous silica nanoprobe for imaging of colonic tumors in vivo.

Hypochlorite acid (ClO-) is one of the major reactive oxygen species (ROS) in colon cancer, providing an effective target for colonic tumor in vivo imaging. For detection of ClO- and tumor imaging, poly[(9,9-di(2-ethylhexyl)-9H-fluorene-2,7-vinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (PFV-co-MEHPV, namely CP1) was encapsulated in mesoporous silica nanoparticles (MSNs) that were pre-modified with polyphenylenevinylene (PPV) via in situ polymerization to construct bright PPV@MSN-CP1 nanoparticles. The synthesized nanoparticles were size-stable and not cytotoxic as confirmed by FE-TEM, FE-SEM, and MTT assay. Hypochlorite oxidizes the vinylidene bond of CP1 through π2-π2 cycloaddition to form PPV-dioxetane intermediates to generate photons. The CL quantum yield of PPV@MSN-CP1 was 16.7 times higher than that of Pluronic F-127 wrapped CP1. CL nanoparticles PPV@MSN-CP1 have good selectivity for hypochlorite detection among biological oxidants (mainly ROS). The linear range and the LOD of PPV@MSN@CP1 for ClO- detection are 4-90 and 1.02 µM, respectively. Subsequently, we further coated PPV@MSN@CP1 with folic acid for tumor targeting by phospholipid wrapping. PPV@MSN-CP1@FA was successfully applied for in vivo imaging of endogenously produced ClO- of tumor tissue in living animals.

Chemiluminescence of Conjugated-Polymer Nanoparticles by Direct Oxidation with Hypochlorite.

Chemiluminescence (CL) is an advantageous detection tool for in vivo imaging because of the high signal-to-noise ratio of its optical-signal readout, which does not require an external excitation source. Conjugated polymers (CPs) are now used as an energy acceptor in CL nanoparticles to enhance the CL. Here, we demonstrate CL from the direct oxidation of CP backbones in conjugated-polymer nanoparticles (CPNs) by hypochlorite. Such CL CPNs completely avoid the involvement of small-molecule CL donors. The strategy greatly simplifies CL-probes preparation and increases the stability of CL nanoprobes by overcoming the leakage problem of CL donors in nanoparticles. Hypochlorite can oxidize the vinylene bond (C═C) in polyfluorene-vinylene (PFV)/polyphenylenevinylene (PPV) via π2-π2 cycloaddition to form a PFV- or PPV-dioxetane intermediate that is unstable and can spontaneously degrade into PFV- or PPV-aldehyde and generate photons. The dioxetane-intermediate formation was confirmed by UV-vis-absorption, fluorescence, nuclear-magnetic-resonance (1H NMR), and Fourier-transform infrared (FT-IR) spectroscopy. The CL quantum yield (QY) of the brightest CL probe, CPN-poly[(9,9-di(2-ethylhexyl)-9 H-fluorene-2,7-vinylene)- co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (90:10 mol ratio, CPN-PFV- co-MEHPV), was 17.79 einsteins/mol (namely, photons per particle). CPN-PFV- co-MEHPV was size-stable, noncytotoxic, selective, and sensitive for hypochlorite detection. The linear range and the LOD of CPN-PFV- co-MEHPV for ClO- detection are 2-30 and 0.47 µM. Thus, CPN-PFV- co-MEHPV was successfully applied for in vivo imaging of endogenously produced ClO- in living animals. We expect that the represented strategy could be extended to construct other CL nanoprobes for bioimaging and disease diagnosis by simply optimizing and transforming CP backbones; such CL CPNs will have a profound impact on the field of bioimaging.

Multifunctional Probe Based on Cationic Conjugated Polymers for Nitroreductase-Related Analysis: Sensing, Hypoxia Diagnosis, and Imaging.

Nitroreductase (NTR) is overexpressed in hypoxic tumors. Moreover, hypoxia is usually considered as the most important feature of various diseases. Thus, it is important to build a sensitive and selective method for NTR detection and hypoxia diagnosis. Herein, a new cationic conjugated polymer (PBFBT-NP) with p-nitrophenyl group in the side chain was designed and synthesized as a fluorescent probe for the detection of NTR. In the absence of NTR, the fluorescence of PBFBT-NP was quenched due to photoinduced electron transfer (PET). On the contrary, in the presence of NTR, NTR can specifically react with p-nitrophenyl group to form p-aminophenyl group, which leads to the PET being inhibited and the polymer's fluorescence significantly increasing (>110-fold). The sensitive and selective NTR sensing method in vitro is thus constructed with a low detection limit of 2.9 ng/mL. Moreover, the hypoxic status of tumor cells can be visualized by fluorescence bioimaging with very low cytotoxicity. Interestingly, the probe was successfully used for imaging an NTR-expressed microorganism, such as E. coli, and showed excellent antibacterial activity against E. coli under white light irradiation. In brief, this multifunctional probe is promising for widespread use in NTR-related biological analysis.

Electrospun fibrous scaffolds promote breast cancer cell alignment and epithelial-mesenchymal transition.

In this work we created electrospun fibrous scaffolds with random and aligned fiber orientations in order to mimic the three-dimensional structure of the natural extracellular matrix (ECM). The rigidity and topography of the ECM environment have been reported to alter cancer cell behavior. However, the complexity of the in vivo system makes it difficult to isolate and study such extracellular topographical cues that trigger cancer cells' response. Breast cancer cells were cultured on these fibrous scaffolds for 3-5 days. The cells showed elongated spindle-like morphology in the aligned fibers, whereas they maintained a mostly flat stellar shape in the random fibers. Gene expression profiling of these cells post seeding showed up-regulation of transforming growth factor ß-1 (TGFß-1) along with other mesenchymal biomarkers, suggesting that these cells undergo epithelial-mesenchymal transitions in response to the polymer scaffold. The results of this study indicate that the topographical cue may play a significant role in tumor progression.

Cationic conjugated polymers for optical detection of DNA methylation, lesions, and single nucleotide polymorphisms.

Simple, rapid, and sensitive technologies to detect nucleic acid modifications have important applications in genetic analysis, clinical diagnosis, and molecular biology. Because genetic modifications such as single nucleotide polymorphisms (SNP), DNA methylation, and other lesions can serve as hallmarks of human disease, interest in such methods has increased in recent years. This Account describes a new strategy for the optical detection of these DNA targets using cationic conjugated polymers (CCPs). Because of their unique signal amplification properties, researchers have extensively investigated conjugated polymers as optical transducers in highly sensitive biosensors. Recently, we have shown that cationic polyfluorene can detect SNPs within the DNA of clinical samples. When we incorporated deoxyguanosine triphosphate (dGTP-Fl) into the DNA chain at an SNP site where the target/probe pair is complementary, we observed higher fluorescence resonance energy transfer (FRET) efficiency between cationic polyfluorene and fluorescein label on the dGTP. By monitoring the change in emission intensity of cationic polyfluorene or fluorescein, we identified the homozygous or heterozygous SNP. The high sensitivity of this assay results from the 10-fold enhancement of fluorescein emission intensity by the FRET from polyfluorene. This method can detect allele frequencies (the proportion of all copies of a gene that is made up of a particular gene variant) as low as 2%. Using this novel method, we clearly discriminated among the SNP genotypes of 76 individuals of Chinese ancestry. Improving on this initial system, we designed a method for multicolor and one-tube SNP genotyping assays based on cationic polyfluorene using fluorescein-labeled deoxyuridine triphosphate (dUTP-Fl) and Cy3-labeled deoxycytidine triphosphate (dCTP-Cy3) in extension reactions. We also developed a one-step method for direct detection of SNP genotypes from genomic DNA by combining allele-specific PCR with CCPs. In 2008, we developed a new method for DNA methylation detection based on single base extension reaction and CCPs. Treatment of DNA with bisulfite followed by PCR amplification converts unmethylated DNA into a C/T polymorphism, which allows us to characterize the methylation status of the target DNA. Furthermore, we used CCPs to detect DNA lesions caused by ultraviolet light irradiation for the first time. By monitoring the color change of cationic polythiophene before and after DNA cleavage, we also detected oxidative damage to DNA by hydroxyl radical. These CCP-based new assays avoid primer labeling, cumbersome workups, and sophisticated instruments, leading to simpler procedures and improved sensitivity. We expect that these features could lead to major advances in human disease diagnostics and genomic study in the near future.

Fluorescent DNA-poly(phenylenevinylene) hybrid hydrogels for monitoring drug release.

A new class of fluorescent DNA-poly(phenylenevinylene) hybrid hydrogels (DNA/SP-PPV) have been prepared by in situ polymerization of monomer with salmon DNA as template, which are used for monitoring drug release driven by media pH.

Fluorescence logic-signal-based multiplex detection of nucleases with the assembly of a cationic conjugated polymer and branched DNA.

An energy-transfer cascade is generated from a cationic conjugated polymer (PFP) and negatively charged, Y-shaped DNA labeled with three dyes at its termini (fluorescein (Fl), Tex Red, and Cy5). Multistep fluorescence resonance energy transfer regulates the fluorescence intensities of PFP and the dyes. Different types of logic gates can be operated by observing the emission wavelengths of different dyes with multiplex nucleases as inputs.

Conjugated polyelectrolyte-DNA complexes for multi-color and one-tube SNP genotyping assays.

The complexes of a cationic conjugated polymer with DNA are designed as new platforms for homogeneous, sensitive and facile fluorescence assays for SNP genotyping, which interface with single-base extension, multi-step FRET and optical amplification properties of conjugated polymers.

Microorganism-based assemblies of luminescent conjugated polyelectrolytes.

A novel approach was developed for the assembly of fluorescent conjugated polyelectrolytes into tubes on the micrometre scale of tunable length using fungi as living templates.

Expansion of breast cancer stem cells with fibrous scaffolds.

Cancer stem cells (CSCs) are hypothesized as tumor-initiating cells within tumors and main contributors of tumor growth, metastasis and recurrence. Mammary cancer cells, MCF-7 cells, were cultured on 3D polycaprolactone (PCL) fibrous scaffolds, showing an increased proportion of CSCs. The expression of stem cell markers, including OCT3/4 and SOX2, and breast CSC-specific markers, SOX4 and CD49f, was significantly upregulated, and the mammosphere-forming capability in cells cultured on PCL fibrous scaffolds increased. The fibrous scaffolds also induced the elongation of MCF-7 cells and extended cell proliferation. The increase of CSC properties after being cultured on fibrous scaffolds was further confirmed with two luminal-type mammary cell lines, T47D and SK-BR-3, and a basal-type cell line, MDA-MB-231, by ALDEFLUOR assay and mammosphere formation assay. Moreover, we observed the upregulation of epithelial to mesenchymal transition and increased invasive capability in cells cultured on PCL fibrous scaffolds. These data suggest that the increase of CSC proportion in a 3D culture system may account for the enhanced malignancy. Therefore, our PCL fibrous scaffolds can potentially be used for CSCs enrichment and anti-cancer drug screening.

A water-soluble conjugated polymer for protein identification and denaturation detection.

Rapid and sensitive methods to detect proteins and protein denaturation have become increasingly needful in the field of proteomics, medical diagnostics, and biology. In this paper, we have reported the synthesis of a new cationic water-soluble conjugated polymer that contains fluorene and diene moieties in the backbone (PFDE) for protein identification by sensing an array of PFDE solutions in different ionic strengths using the linear discriminant analysis technique (LDA). The PFDE can form complexes with proteins by electrostatic and/or hydrophobic interactions and exhibits different fluorescence response. Three main factors contribute to the fluorescence response of PFDE, namely, the net charge density on the protein surface, the hydrophobic nature of the protein, and the metalloprotein characteristics. The denaturation of proteins can also be detected using PFDE as a fluorescent probe. The interactions between PFDE and proteins were also studied by dynamic light scattering (DLS) and isothermal titration microcalorimetry (ITC) techniques. In contrast to other methods based on conjugated polymers, the synthesis of a series of quencher or dye-labeled acceptors or protein substrates has been avoided in our method, which significantly reduces the cost and the synthetic complexity. Our method provides promising applications on protein identification and denaturation detection in a simple, fast, and label-free manner based on non-specific interaction-induced perturbation of PFDE fluorescence response.

Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution.

This protocol describes a simple, convenient and sensitive single-nucleotide polymorphism (SNP) genotyping method using an optically amplifying cationic conjugated polymer and single base primer extension reaction. The fluorescence resonance energy transfer (FRET) efficiency between the conjugated polymer (PFP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide}) and a fluorescein-labeled dNTP (dNTP-Fl) is correlated to the incorporation of the dNTP-Fl into an allele-specific primer; incorporation occurs by a single base extension reaction when the target DNA and the primer are complementary at the SNP site. By triggering the FRET from PFP to fluorescein and measuring the change in fluorescence intensity of samples, the SNP genotypes can be discriminated. In comparison with other SNP genotyping methods, this protocol simplifies procedures and improves sensitivity by eliminating the need for primer labeling, cumbersome workups, chemical/enzymatic coupling reactions and sophisticated instruments. The assay takes about 2 h for PCR amplification followed by 5.5-7.5 h to obtain the genotypes.

A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels.

A sensitive chemiluminescent (CL) immunoassay of human immunoglobulin (IgG) which combined the inherent high sensitivity of CL analysis with the dramatic signal amplification of silver precipitation on colloidal gold tags was developed. First, the sandwich-type complex was formed in this protocol by the primary antibody immobilized on the polystyrene wells, the analyte in the sample, and the secondary antibody labeled with colloidal gold. Second, the colloidal gold was treated by an Ag(+) reduction solution, which resulted in the catalytic precipitation of silver on the surface of colloidal gold. Third, a large number of Ag(+) were oxidatively released in HNO(3) solution from the silver metal anchored on the sandwich-type complexes and then the human IgG was indirectly determined by a sensitive combined CL reaction of Ag(+)-K(2)S(2)O(8)-Mn(2+)- H(3)PO(4)-luminol. The chemiluminescence intensity depends linearly on the logarithm of the concentration of human IgG over the range of 0.02-50ngml(-1) and detection limit (3sigma) is 0.005ngml(-1) (i.e., approximately 3x10(-14)M, 3amol in 100-mul sample). This assay has been successfully applied to the determination of human IgG in human serum samples and showed great potential for numerous applications in immunoassay.