One-Step Polymerase Chain Reaction-Free Nanowire-Based Plasma Cell-Free DNA Assay to Detect EML4-ALK Fusion and to Monitor Resistance in Lung Cancer.

BACKGROUND: Next-generation sequencing has mostly been used for genotyping cell-free DNA (cfDNA) in plasma. However, this assay has several clinical limitations. We evaluated the clinical utility of a novel polymerase chain reaction-free nanowire (NW)-based plasma cfDNA assay for detecting ALK fusion and mutations. PATIENTS, MATERIALS, AND METHODS: We consecutively enrolled 99 patients with advanced non-small cell lung cancer undergoing a fluorescence in situ hybridization (FISH) test for ALK fusion; ALK-positive (n = 36). The NW-based assay was performed using 50-100 µL of plasma collected at pretreatment and every 8 weeks during ALK inhibitor treatment. RESULTS: There was high concordance between the NW-based assay and the FISH test for identification of ALK fusion (94.9% with a kappa coefficient value of 0.892, 95% confidence interval [CI], 0.799-0.984). There was no difference in the response rate to the first anaplastic lymphoma kinase inhibitor between the ALK-positive patients identified by the NW-based assay and by the FISH test (73.5% vs. 72.2%, p = .931). In the ALK variant analysis, variants 1 and 3 subgroups were detected in 27 (75.0%) and 8 (22.2%) patients, respectively. Among 24 patients treated with crizotinib, variant 3 subgroup was associated with worse median overall survival than variant 1 subgroup (36.5 months; 95% CI, 0.09-87.6 vs. 19.8 months; 95% CI, 9.9-not reached, p = .004]. A serial assessment identified that ALK L1196M resistance mutation emerged before radiologic progression during crizotinib treatment. CONCLUSION: The newly developed simple NW-based cfDNA assay may be clinically applicable for rapid diagnosis of ALK fusion with its variant forms and early detection of resistance. IMPLICATIONS FOR PRACTICE: The authors developed a novel one-step polymerase chain reaction-free nanowire (NW)-based plasma cell-free DNA (cfDNA) assay. This study evaluated the clinical utility of this novel method for the diagnosis of EML4-ALK fusion in advanced non-small cell lung cancer (NSCLC). The NW-based assay and FISH test showed high concordance rate in 99 patients with advanced NSCLC. Serial cfDNA assessment demonstrated this method provided early detection of resistance before radiologic progression during crizotinib treatment. Taken together, plasma cfDNA genotyping by the NW-based cfDNA assay may be useful for the rapid diagnosis of ALK fusion, classifying variants, and early detection of resistance.

Assessment of clinical performance of an ultrasensitive nanowire assay for detecting human papillomavirus DNA in urine.

OBJECTIVE: To evaluate whether HPV DNA in urine has potential advantages as an alternative biomarker for HPV-based cervical cancer screening. METHODS: Among patients with Cobas HPV test results, a total of 67 HPV-positive (n = 42) and -negative (n = 25) women who agreed to participate in this study were willing to provide paired cervical and urine samples, and we observed concordance between sample types from each patient in identifying HPV genotypes using the nanowire assay. RESULTS: We detected high-risk strains of HPV DNA in unprocessed urine specimens using polyethyleneimine-conjugated nanowires (PEI-NWs). Concordance for high-risk HPV (hrHPV) between paired urine and cervical samples was 90.4% (κ = 0.90; 95% CI: 0.80-100.00). The virological sensitivity and specificity for detection of HPV DNA from a small urine sample (200 µL) were 81.3% (κ = 0.83; 95% CI: 62.1-100.0) and 98.0% (κ = 0.83; 95% CI: 94.2-100.0) for HPV16 group, 100.0% (κ = 0.65; 95% CI: 100.0-100.0) and 95.3% (κ = 0.65; 95% CI: 90.1-100.0) for HPV18 group, and 96.4% (κ = 0.97; 95% CI: 89.6-100.0) and 100.0% (κ = 0.97; 95% CI: 100.0-100.0) for other hrHPV group, respectively. CONCLUSIONS: The nanowire assay demonstrated excellent ability to identify HPV DNA from urine specimens. We observed an excellent agreement in the detection of high-risk HPV between paired urine and cervical samples, even with small urine sample volume.

Direct isolation and characterization of circulating exosomes from biological samples using magnetic nanowires.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Improved biosynthesis of silver nanoparticles using keratinase from Stenotrophomonas maltophilia R13: reaction optimization, structural characterization, and biomedical activity.

In the present study, keratinase from Stenotrophomonas maltophilia R13 was used for the first time as a reducing agent for the eco-friendly synthesis of AgNPs. The keratinase produced by strain R13 was responsible for the reduction of silver ions and the subsequent formation of AgNPs. Maximum AgNP synthesis was achieved using 2 mM AgNO3 at pH 9 and 40 °C. Electron microscopy and dynamic light scattering analysis showed AgNPs were spherical and of average diameter ~ 8.4 nm. X-ray diffraction revealed that AgNPs were crystalline. FTIR indicated AgNPs were stabilized by proteins present in the crude enzyme solution of strain R13. AgNPs exhibited a broad antimicrobial spectrum against several pathogenic microorganisms, and the antimicrobial mechanism appeared to involve structural deformation of cells resulting in membrane leakage and subsequent lysis. AgNPs also displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 0.0112 mg/ml), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate radical scavenging (IC50 = 0.0243 mg/ml), and anti-collagenase (IC50 = 23.5 mg/ml) activities.

Predictive value of circulating tumor cells (CTCs) captured by microfluidic device in patients with epithelial ovarian cancer.

OBJECTIVE: To test an electrically conductive chip, incorporating a nanoroughened microfluidic platform for the capture of circulating tumor cells (CTCs), and assess its clinical merit in instances of epithelial ovarian cancer (EOC). METHODS: A total of 54 patients with EOC recruited between August 2014 and May 2015 were enrolled in this prospective study. CTCs in peripheral blood were detected in advance of primary tumor resection and before initiating adjuvant chemotherapy for recurrent disease. We identified CTCs as EpCAM-positive and DAPI-positive, and CD45-negative feature. RESULTS: Twenty-four patients with primary disease and 30 patients with recurrences were included in the study. CTCs were detected in 98.1% (53/54). In newly diagnosed patients, median counts of single CTCs and CTC clusters were 4 (0-13) and 1(0-14), respectively. In those with recurrences, median counts were 3 (1-9) and 1(0-24), respectively. Such counts did not differ significantly by tumor stage or by serum CA125 level; but progression-free survival declined at a cutpoint of ≥3 CTCs, and CTC-cluster positivity correlated with platinum resistance. Isolated CTCs (successfully cultured ex vivo in two patients) showed greater sensitivity to anticancer drugs and proliferated more rapidly than did established cell lines. CONCLUSION: Proof-of-concept was provided for an electrically conductive and nanoroughened microfluidic platform-based chip designed to capture CTCs in patients with EOC. A larger patient sampling and longer duration of follow-up are needed to determine its suitability for clinical use.

Electroactive polypyrrole nanowire arrays: synergistic effect of cancer treatment by on-demand drug release and photothermal therapy.

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.

Action at a distance: functional drug delivery using electromagnetic-field-responsive polypyrrole nanowires.

In this work, we introduce a free-standing, vertically aligned conductive polypyrrole (Ppy) architecture that can serve as a high-capacity drug reservoir. This novel geometric organization of Ppy provides a new platform for improving the drug-loading efficiency. Most importantly, we present the first formal evidence that an impregnated drug (dexamethasone, DEX) can be released on demand by a focal, pulsatile electromagnetic field (EMF). This remotely controlled, on-off switchable polymer system provides a framework for implantable constructs that can be placed in critical areas of the body without any physical contact (such as percutaneous electrodes) with the Ppy, contributing to a low "foreign body" footprint. We demonstrate this possibility by using a BV-2 microglia culture model in which reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS) expression was attenuated in response to DEX released from EMF-stimulated Ppy.

An electroactive biotin-doped polypyrrole substrate that immobilizes and releases EpCAM-positive cancer cells.

The specific capture and remotely controlled release of the EpCAM-positive cancer cells from biotin-doped polypyrrole (Ppy) films in response to an electrical potential is presented. As Ppy allows the direct incorporation of biotin molecules during the electrochemical process, densely packed biotin molecules can serve as the binding sites for streptavidin-tagged biomolecular complexes. This study demonstrates not only the enhanced capture and enrichment of EpCAM-positive cancer cells but also "on-demand" release of the viable cells from conductive Ppy in an electrical-potential-dependent way. This novel approach is of great importance in a diverse range of applications, and in particular in cancer diagnostics and screening.

Early On-Treatment Prediction of the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors.

BACKGROUND: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) remains challenging. Thus, we investigated whether resistant cancer cells that expand shortly after EGFR-TKI treatment would eventually cause the resistant phenotype. METHODS: We generated two EGFR-mutant lung cancer cell lines resistant to gefitinib (PC9GR and HCC827GR). The parent cell lines were exposed to short-term treatment with gefitinib or paclitaxel and then were assessed for EGFR T790M mutation and C-MET expression. These experiments were repeated in vivo and in clinically relevant patient-derived cell (PDC) models. For validation in clinical cases, we measured these gene alterations in plasma circulating tumor DNA (ctDNA) before and 8 weeks after starting EGFR-TKIs in four patients with EGFR-mutant lung cancer. RESULTS: T790M mutation was only detected in the PC9GR cells, whereas C-MET amplification was detected in the HCC827GR cells. The T790M mutation level significantly increased in PC9 cells after short-term treatment with gefitinib but not in the paclitaxel. C-MET mRNA expression was only significantly increased in gefitinib-treated HCC827 cells. We confirmed that the C-MET copy number in HCC827 cells that survived after short-term gefitinib treatment was significantly higher than that in dead HCC827 cells. These findings were reproduced in the in vivo and PDC models. An early on-treatment increase in the plasma ctDNA level of these gene alterations was correlated with the corresponding resistance mechanism to EGFR-TKIs, a finding that was confirmed in post-treatment tumor tissues. CONCLUSIONS: Early on-treatment kinetics in resistance-related gene alterations may predict the final mechanism of EGFR-TKI resistance.

Biotin-doped porous polypyrrole films for electrically controlled nanoparticle release.

A novel method for the preparation of biotin-doped porous conductive surfaces has been suggested for a variety of applications, especially for an electrically controlled release system. Well-ordered and three-dimensional porous conductive structures have been obtained by the electrochemical deposition of the aqueous biotin-pyrrole monomer mixture into particle arrays, followed by subsequent removal of the colloidal particles. Advantageously, direct incorporation of biotin molecules enhances the versatility by modifying surfaces through site-directed conjugate formation, thus facilitating further reactions. In addition, the porosity of the surfaces provides a significant impact on enhanced immobilization and efficient release of streptavidin-tagged gold nanoparticles. Biotinylated porous polypyrrole (Ppy) films were characterized by several techniques: (1) scanning electron microscopy (SEM) to evaluate surface topography, (2) X-ray photoelectron spectroscopy (XPS) to assess the potential-dependent chemical composition of the films, (3) four-point probe evaluation to measure the conductivity, cyclic voltammetry to observe surface eletroactivity, and contact angle measurement to evaluate the surface wettability, and (4) fluorescence microscopy to image and quantify the adsorption and release of gold nanoparticles. Overall, our results demonstrate that these biotinylated porous Ppy films, combined with electrical stimulation, permit a programmable release of gold nanoparticles by altering the chemical strength of the Ppy-biotin interaction.

Well-ordered porous conductive polypyrrole as a new platform for neural interfaces.

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.

A nanowire-based liquid biopsy method using cerebrospinal fluid cell-free DNA for targeted management of leptomeningeal carcinomatosis.

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.

Circulating Small Extracellular Vesicles Activate TYRO3 to Drive Cancer Metastasis and Chemoresistance.

Extracellular vesicles (EV) in the tumor microenvironment have emerged as crucial mediators that promote proliferation, metastasis, and chemoresistance. However, the role of circulating small EVs (csEV) in cancer progression remains poorly understood. In this study, we report that csEV facilitate cancer progression and determine its molecular mechanism. csEVs strongly promoted the migration of cancer cells via interaction with phosphatidylserine of csEVs. Among the three TAM receptors, TYRO3, AXL, and MerTK, TYRO3 mainly interacted with csEVs. csEV-mediated TYRO3 activation promoted migration and metastasis via the epithelial-mesenchymal transition and stimulation of RhoA in invasive cancer cells. Additionally, csEV-TYRO3 interaction induced YAP activation, which led to increased cell proliferation and chemoresistance. Combination treatment with gefitinib and KRCT-6j, a selective TYRO3 inhibitor, significantly reduced tumor volume in xenografts implanted with gefitinib-resistant non-small cell lung cancer cells. The results of this study show that TYRO3 activation by csEVs facilitates cancer cell migration and chemoresistance by activation of RhoA or YAP, indicating that the csEV/TYRO3 interaction may serve as a potential therapeutic target for aggressive cancers in the clinic. SIGNIFICANCE: These findings demonstrate that circulating extracellular vesicles are a novel driver in migration and survival of aggressive cancer cells via TYRO3 activation. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3539/F1.large.jpg.

Functional silica nanoparticle-mediated neuronal membrane sealing following traumatic spinal cord injury.

The mechanical damage to neurons and their processes induced by spinal cord injury (SCI) causes a progressive cascade of pathophysiological events beginning with the derangement of ionic equilibrium and collapse of membrane permeability. This leads to a cumulative deterioration of neurons, axons, and the tissue architecture of the cord. We have previously shown that the application of the hydrophilic polymer polyethylene glycol (PEG) following spinal cord or brain injury can rapidly restore membrane integrity, reduce oxidative stress, restore impaired axonal conductivity, and mediate functional recovery in rats, guinea pigs, and dogs. However there are limits to both the concentration and the molecular weight of the application that do not permit the broadest recovery across an injured animal population. In this study, PEG-decorated silica nanoparticles (PSiNPs) sealed cells, as shown by the significantly reduced leakage of lactate dehydrogenase from damaged cells compared with uncoated particles or PEG alone. Further in vivo tests showed that PSiNPs also significantly reduced the formation of reactive oxygen species and the process of lipid peroxidation of the membrane. Fabrication of PSiNPs containing embedded dyes also revealed targeting of the particles to damaged, but not undamaged, spinal cord tissues. In an in vivo crush/contusion model of guinea pig SCI, every animal but one injected with PSiNPs recovered conduction through the cord lesion, whereas none of the control animals did. These findings suggest that the use of multifunctional nanoparticles may offer a novel treatment approach for spinal cord injury, traumatic brain injury, and possibly neurodegenerative disorders.

Chitosan produces potent neuroprotection and physiological recovery following traumatic spinal cord injury.

Chitosan, a non-toxic biodegradable polycationic polymer with low immunogenicity, has been extensively investigated in various biomedical applications. In this work, chitosan has been demonstrated to seal compromised nerve cell membranes thus serving as a potent neuroprotector following acute spinal cord trauma. Topical application of chitosan after complete transection or compression of the guinea pig spinal cord facilitated sealing of neuronal membranes in ex vivo tests, and restored the conduction of nerve impulses through the length of spinal cords in vivo, using somatosensory evoked potential recordings. Moreover, chitosan preferentially targeted damaged tissues, served as a suppressor of reactive oxygen species (free radical) generation, and the resultant lipid peroxidation of membranes, as shown in ex vivo spinal cord samples. These findings suggest a novel medical approach to reduce the catastrophic loss of behavior after acute spinal cord and brain injury.

The preparation of polypyrrole surfaces in the presence of mesoporous silica nanoparticles and their biomedical applications.

The deposition of carboxylic acid-terminated conducting polymer into two- or three-dimensional structures made up of colloidal particles has been successfully completed. This was accomplished using electrochemical deposition of ordered arrays of mesoporous silica nanoparticles (MSNs) as a template. Subsequent removal of the template yielded a porous polypyrrole surface. The co-polymerization of pyrrole with carboxylic acid-terminated pyrrole derivatives overcame the limitations of a lack of reactive functional groups--by facilitating the direct coupling of the film with biomolecules or drugs on the surface. Such Ppy films were characterized by several techniques: (1) scanning electron microscope (SEM) to evaluate surface topography, (2) x-ray photoelectron spectroscopy (XPS) to assess the chemical composition of the films, (3) four-point probe to measure the conductivity, and cyclic voltammogram to observe surface electroactivity. To assay the biological effectiveness of this preparation, we used phase-contrast light microscopy to compare neurite outgrowth from PC 12 cells grown on Ppy films in the presence and absence of electrical stimulation. These electrically functional, biocompatible composites show promise as novel neural implants that would deliver specific biologically active molecules in a highly localized manner to damaged or otherwise vulnerable cells such as found in the nervous system.

A mesoporous silica nanosphere-based drug delivery system using an electrically conducting polymer.

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.

A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine.

Purpose: As human papillomavirus (HPV) is primarily responsible for the development of cervical cancer, significant efforts have been devoted to develop novel strategies for detecting and identifying HPV DNA in urine. The analysis of target DNA sequences in urine offers a potential alternative to conventional methods as a non-invasive clinical screening and diagnostic assessment tool for the detection of HPV. However, the lack of efficient approaches to isolate and directly detect HPV DNA in urine has restricted its potential clinical use. In this study, we demonstrated a novel approach of using polyethylenimine-conjugated magnetic polypyrrole nanowires (PEI-mPpy NWs) for the extraction, identification, and PCR-free colorimetric detection of high-risk strains of HPV DNA sequences, particularly HPV-16 and HPV-18, in urine specimens of cervical cancer patients. Materials and Methods: We fabricated and characterized polyethylenimine-conjugated magnetic nanowires (PEI/mPpy NWs). PEI/mPpy NWs-based HPV DNA isolation and detection strategy appears to be a cost-effective and practical technology with greater sensitivity and accuracy than other urine-based methods. Results: The analytical and clinical performance of PEI-mPpy NWs was evaluated and compared with those of cervical swabs, demonstrating a superior type-specific concordance rate of 100% between urine and cervical swabs, even when using a small volume of urine (300 µL). Conclusion: We envision that PEI-mPpy NWs provide substantive evidence for clinical diagnosis and management of HPV-associated disease with their excellent performance in the recovery and detection of HPV DNA from minimal amounts of urine samples.

A Novel Multifunctional Nanowire Platform for Highly Efficient Isolation and Analysis of Circulating Tumor-Specific Markers.

Circulating tumor-specific markers are crucial to understand the molecular and cellular processes underlying cancer, and to develop therapeutic strategies for the treatment of the disease in clinical applications. Many approaches to isolate and analyze these markers have been reported. Here, we propose a straightforward method for highly efficient capture and release of exosomes and circulating tumor cells (CTCs) in a single platform with well-ordered three-dimensional (3D) architecture that is constructed using a simple electrochemical method. Conductive polypyrrole nanowires (Ppy NWs) are conjugated with monoclonal antibodies that specifically recognize marker proteins on the surface of exosomes or CTCs. In response to electrical- or glutathione (GSH)-mediated stimulation, the captured exosomes or cells can be finely controlled for retrieval from the NW platform. A surface having nano-topographic structures allows the specific recognition and capture of small-sized exosome-like vesicles (30-100 nm) by promoting topographical interactions, while physically blocking larger vesicles (i.e., microvesicles, 100-1,000 nm). In addition, vertically aligned features greatly improve cell capture efficiency after modification with desired high-binding affinity biomolecules. Notably, exosomes and CTCs can be sequentially isolated from cancer patients' blood samples using a single NW platform via modulating electrochemical and chemical cues, which clearly exhibits great potential for the diagnosis of various cancer types and for downstream analysis due to its facile, effective, and low-cost performance.

Magnetic Nanowire Networks for Dual-Isolation and Detection of Tumor-Associated Circulating Biomarkers.

Purpose: 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. Materials and Methods: 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 EGFR mutations based on digital droplet PCR analysis of cfDNA and CTC DNA from patients with lung cancer. Results: 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. Conclusion: The simple, versatile, and highly efficient nanowire network tool allows sensitive and robust assessment of clinical samples.