Abstract Circulating tumor cells (CTCs) are recognized as promising biomarkers for diagnosis and indication of the prognosis of several epithelial cancers. However, at present, CTC monitoring is ...available only for advanced-stage patients rather than for those at an early stage of cancer. This is because of the extraordinary rarity of CTCs and the limited sensitivity of current methods. Herein, we report the development of multifunctional magnetic nanowires for the efficient isolation and detection of CTCs from the blood of patients, especially those with non-metastatic early-stage cancer. The nanowires, which are equipped with a high density of magnetic nanoparticles and five different types of antibodies (Ab mixture_mPpyNWs), offer a significant improvement in cell-isolation efficiency, even from very small amounts of blood (250 μL–1 mL). Notably, CTCs were isolated and identified in 29 out of 29 patients (100%) with non-metastatic early breast cancer, indicating that this procedure allowed detection of CTCs with greater accuracy, sensitivity, and specificity. In addition, we demonstrated in situ “naked eye” identification of the captured cancer cells via a simple colorimetric immunoassay. Our results show that antibody-functionalized magnetic nanowires offer great potential for a broad range of practical clinical applications, including early detection, diagnosis, and treatment of cancer.
Tumor-derived exosomes are gaining attention as important factors that facilitate communication between neighboring cells and manipulate cellular processes associated with cancer development or ...progression. The conventional techniques for the isolation and detection of exosomes face several limitations, restricting their clinical applications. Hence, a highly efficient technique for the isolation and identification of exosomes from biological samples may provide critical information about exosomes as biomarkers and improve our understanding of their unique role in cancer research. Here, we describe the use of antibody cocktail-conjugated magnetic nanowires to isolate exosomes from plasma of breast and lung cancer patients.
The isolated exosomes were characterized based on size and concentration using nanoparticle tracking analysis. Levels of exosomal proteins were measured by bicinchoninic acid assay and enzyme-linked immunosorbent assay. Morphology was visualized by transmission electron microscopy. Immunoblotting (Western blotting) was used to detect the presence of exosomal markers.
The use of antibody cocktail-conjugated magnetic nanowires resulted in approximately threefold greater yield when compared to the conventional methods. The elongated feature of nanowires significantly improved the efficiency of exosome isolation, suggesting its potential to be translated in diverse clinical applications, including cancer diagnosis and treatment.
The nanowire-based method allows rapid isolation of homogeneous population of exosomes with relatively high yield and purity from even small amounts of sample. These results suggest that this method has the potential for clinical applications requiring highly purified exosomes for the analysis of protein, lipid, mRNA, and miRNA.
We have suggested a novel method for the preparation of a label-free electrochemical immunosensor for the detection of prostate-specific antigen (PSA) as target marker for prostate cancer. Direct ...incorporation of PSA antibody (anti-PSA) into polypyrrole (Ppy) electropolymerized on a three-dimensional Au nanowire array has resulted in enhanced molecular interactions, ultimately leading to improved sensing performance. The electrochemical performance of the nanowire-based immunosensor array were characterized by (1) differential pulse voltammetry (DPV) to evaluate the specific recognition of PSA, (2) impedance and cyclic voltammetry to observe surface resistance and electroactivity, and (3) scanning electron microscopy (SEM) to demonstrate the three-dimensional architecture. The vertically-aligned geometric organization of Ppy provides a novel platform to improve the anti-PSA loading capacity. Overall, enhanced electrochemical performance of the proposed immunosensor has been demonstrated by its linear response over PSA concentrations ranging from 10fgmL−1 to 10ngmL−1 and a detection limit of 0.3fgmL−1, indicating that the strategy proposed here has great potential for clinical applications.
•Polypyrrole (Ppy) nanowire immunosensor was developed for the detection of PSA.•Direct incorporation of anti-PSA into Ppy affords enhanced molecular interactions.•Vertically-aligned geometry of Ppy improves anti-PSA loading efficiency.•The proposed immunosensor demonstrated specific and sensitive detection capability.
Abstract Here, we propose an integrated multifunctional system constructed by conductive disulfide-biotin-doped polypyrrole nanowires (SS-biotin-Ppy NWs) for capture, release, and in situ ...quantification of circulating tumor cells (CTCs). A well-ordered three-dimensional nanowire structure equipped with a monoclonal antibody offers a significant impact on the cell-capture efficiency, as well as on electrical- or glutathione (GSH)-mediated release of the captured cells. In addition, the electrochemical identification/detection of the captured cancer cells can be directly conducted on the same Ppy NW platform by using horseradish peroxidase (HRP)-labeled and anti-EpCAM-conjugated nanoparticles (HRP/anti-EpCAM Ppy NPs), showing very high sensitivity and specificity. The signal amplification can be clearly attributed to the catalytic response resulting from enzymatic reduction of hydrogen peroxide on Ppy NWs, consequently generating a greatly increased amperometric response with a detection range of 10 to 1 × 104 cells and a detection limit of as low as 10 cells. Overall, the proposed Ppy NWs not only present a promising platform for effective cell capture and release but also permit cytosensing capability for on-site analysis.
An electroresponsive drug release system based on polypyrrole (Ppy) nanowires was developed to induce the local delivery of anticancer drug, doxorubicin (DOX), according to the applied electric ...field. DOX-conjugated Ppy nanowire (NW) (DOX/Ppy NW) array was initially prepared by electrochemical deposition of a mixture of pyrrole monomers and biotin as dopants in the anodic alumina oxide membrane as a sacrificial template. Morphological observation by scanning electron microscopy revealed free-standing and 3D nanotopographical features with large surface area and high density. In addition, we investigated the antitumor efficacy of DOX released from DOX/Ppy NW array in response to the external electric field using two kinds of cancer cell lines, human oral squamous carcinoma cells (KB cells) and human breast cancer cells (MCF7 cells). Meanwhile, strong photothermal effect as a result of a near-infrared absorbing ability of Ppy synergistically maximizes the chemotherapeutic efficacy. Our results suggested that the proposed multifunctional Ppy platform possessing several beneficial features is very promising for many therapeutic applications including cancer.
We present the preparation of electrically conductive, porous polypyrrole surfaces and demonstrate their use as an interactive substrate for neuronal growth. Nerve growth factor (NGF)-loaded porous ...conducting polymers were initially prepared by electrochemical deposition of a mixture of pyrrole monomers and NGF into two- or three-dimensional particle arrays followed by subsequent removal of a sacrificial template. Morphological observation by scanning electron microscopy (SEM) revealed these to possess high regularity and porosity with well-defined topographical features. A four-point probe study demonstrated remarkable electrical activities despite the presence of voids. In addition, we investigated the effects of these surfaces on cellular behaviors using PC 12 cells in the presence and absence of electrical stimulation. Our results suggest that the surface topography as well as an applied electrical field can play a crucial role in determining further cell responses. Indeed, surface-induced preferential regulation leads to enhanced cellular viability and neurite extension. Establishing the underlying cellular mechanisms in response to various external stimuli is essential in that one can elicit positive neuronal guidance and modulate their activities by engineering a series of electrical, chemical, and topographical cues.
Purpose
This study aimed to evaluate whether genotyping cell free DNA (cfDNA) in the cerebrospinal fluid (CSF) may be helpful in managing leptomeningeal carcinomatosis (LMC) of
EGFR
-mutant non-small ...cell lung cancer (NSCLC).
Methods
Patients with
EGFR
-mutant NSCLC who progressed as LMC after 3rd-generation tyrosine kinase inhibitors (EGFR–TKIs) were evaluated. A nanowire-based cfDNA assay was performed for genotyping cfDNA from the CSF and plasma. We focused on de novo
EGFR
C797S mutation and
MET
amplification, which are the most common mechanisms of resistance to 3rd-generation EGFR–TKIs.
Results
Among 11 patients, five (45.5%) had progression only at the leptomeninges. The tumor-associated CSF-cfDNA was identified in eight (72.7%) patients, and plasma-cfDNA in six (54.5%) patients. In the CSF-cfDNA,
EGFR
C797S mutation and
MET
amplification were detected in four (36.3%) and two (18.2%) patients, respectively. Of four patients with the C797S-positive LMC, only one had concurrent CSF-T790M mutation. Three patients who had the C797S-positive LMC without CSF-T790M mutation, received 1st–2nd generation EGFR–TKIs and showed clinical benefits for 20.8, 17.8, and 8.8 weeks, respectively. Serial assessment with cfDNA in these patients demonstrated that the CSF levels of C797S mutation were decreased with radiological or neurological improvement but the plasma levels of T790M mutation were markedly increased before objective progression.
Conclusion
Genotyping CSF-cfDNA by the nanowire-based assay is feasible and effective in guiding the treatment of LMC in patients with
EGFR
-mutant NSCLC.
Detecting human papillomavirus (HPV) is central in diagnosing and monitoring HPV-related disease. However, limited sensitivity and the wide variability of the HPV genome pose challenges in the ...identification of HPV genes, particularly high-risk types. This study reports the development of polyethyleneimine-conjugated magnetic nanowires (PEI-MNWs) and their use in the isolation, identification, and analysis of multiple genotypes of HPV DNA from cervical cancer specimens. The nanowires are electrochemically doped with a high density of magnetic nanoparticles and biotin moieties during potentiostatic deposition, thereby allowing conjugating cationic branched polymers to direct the attachment of negatively charged DNA molecules with strong magnetic response. For proof of concept, the rapid and ultrasensitive isolation of HPV DNA is performed at concentrations as low as 10pg/mL with an efficiency of >95%. For clinical optimization, the analytical and clinical sensitivity of PEI-MNWs is compared with that of the Roche Cobas 4800 HPV Test and demonstrates excellent correlation for multiple HPV DNA genotypes with superior threshold cycle values. The high sensitivity, specificity, and good reproducibility of PEI-MNWs are particularly well suited for the recovery of DNA and provide significant and clinically meaningful evidence for the early detection and treatment of HPV-associated cancers.
•The magnetic nanowires were electrochemically developed via potentiostatic deposition.•Nanowires enhance the attachment of polymer chains that ultimately improve DNA recovery.•Nanowires demonstrated the isolation and analysis of multiple genotypes of HPV DNA.•HPV DNA detection and genotyping was confirmed for analytical and clinical validation.
Recent developments in genomic and molecular methods have revolutionized the range of utilities of tumor-associated circulating biomarkers in both basic and clinical research. Herein, we present a ...novel approach for ultrasensitive extraction of cfDNA and CTCs, at high yield and purity, via the formation of magnetic nanowire networks.
We fabricated and characterized biotinylated cationic polyethylenimine and biotinylated antibody cocktail-conjugated magnetic polypyrrole NWs (PEI/mPpy NW and Ab cocktail/mPpy NW, respectively). We applied these NWs to the extraction of cfDNA and CTC from the blood of 14 patients with lung cancer. We demonstrated reliable detection of
mutations based on digital droplet PCR analysis of cfDNA and CTC DNA from patients with lung cancer.
The NW networks confined with a high density of magnetic nanoparticles exhibited superior saturation magnetization, which enabled rapid and high-yield capture whilst avoiding or minimizing damage and loss. The NW networks enabled the co-isolation of CTCs and cfDNA of high quality and sufficient quantities, thus allowing the amplification of rare and low-prevalence cancer-related mutations.
The simple, versatile, and highly efficient nanowire network tool allows sensitive and robust assessment of clinical samples.
In this study, a mesoporous silica nanoparticle (MSN)-based nerve growth factor (NGF) delivery system has been successfully embedded within an electroactive polypyrrol (Ppy). The spherical particles ...with approximately 100 nm diameter possess a large surface-to-volume ratio for the entrapment of NGF into the pores of MSNs while retaining their bioactivity. Direct incorporation of MSN-NGF within Ppy was achieved during electrochemical polymerization. The loading amount and release profile of NGF from the composite was investigated by sandwich ELISA. The NGF incorporation can be controllable by varying particle concentration or by extending electrodeposition time. The morphology and chemical composition of the Ppy/MSN-NGF composite was evaluated by atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and x-ray photoelectron spectroscopy (XPS). Optical and electron microscopy revealed a characteristic attachment of PC 12 cells and the outgrowth of their neurites when grown on the Ppy/MSN-NGF composite as a result of a sustained and controlled release of NGF. In order to observe the effectiveness of electrical stimulation, neurite extension of cells cultured on unstimulated and stimulated Ppy/MSN-NGF was compared. The NGF release in the presence of electrical stimulation promoted significantly greater neurite extension.
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