Multifunctional magnetic nanowires: A novel breakthrough for ultrasensitive detection and isolation of rare cancer cells from non-metastatic early breast cancer patients using small volumes of blood.

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.

Dual-responsive immunosensor that combines colorimetric recognition and electrochemical response for ultrasensitive detection of cancer biomarkers.

We developed a nanoroughened, biotin-doped polypyrrole immunosensor for the detection of tumor markers through dual-signal (electrochemical and colorimetric) channels, electrochemical and colorimetric, that demonstrates remarkable analytical performance. A rapid, one-step electric field-mediated method was employed to fabricate the immunosensor with nanoscale roughness by simply modulating the applied electrical potential. We demonstrated the successful detection of three tumor markers (CA125, CEA, and PSA) via the double enzymatic signal amplifications in the presence of a target antigen, ultimately leading to desired diagnostic accuracy and reliability. The addition of multiple horseradish peroxidase (HRP)- and antibody-labeled nanoparticles greatly amplified the signal and simplified the measurement of cancer biomarker proteins by sequentially magnifying electrochemical and colorimetric signals in a single platform. The two parallel assays performed using the proposed immunosensor have yielded highly consistent and reproducible results. Additionally, for the analysis of plasma samples in a clinical setting, the values obtained with our immunosensor were validated by correlating the results with those of a standard radioimmunoassay (RIA), which obtained very similar clinically valid responses.

Direct Tracking of Amyloid and Tu Dynamics in Neuroblastoma Cells Using Nanoplasmonic Fiber Tip Probes.

Amyloid plaques and neurofibrillary tangles are the pathological hallmarks of Alzheimer's disease. However, there has been a long-standing discussion on the dynamic relations between Aß and tau proteins, partially due to the lack of a tool to track protein dynamics in individual live neurons at the early stage of Aß generation and tau phosphorylation. Here, we developed nanoplasmonic fiber tip probe (nFTP) technology to simultaneously monitor Aß42 generation and tau phosphorylation (at serine 262) in living, single neuroblastoma cells over 12 h. We observed that Aß42 generation, under clinically relevant anesthetic treatment, preceded tau phosphorylation, which then facilitated Aß42 generation. This observation is also supported by measuring proteins in cell lysates using the ultrasensitive label-free photonic crystal nanosensors. nFTP therefore provides an advanced method to investigate protein expression and post-translational modification in live cells and determine outcomes of intervention of Alzheimer's disease and other neurodegenerative disorders.

A reusable electrochemical immunosensor fabricated using a temperature-responsive polymer for cancer biomarker proteins.

In the present study, we describe a reusable electrochemical immunosensor for the repeated detection of cancer biomarkers using a single platform. The integration of a temperature-responsive polymer on the electrode surface enables easy manipulation of the biological sensing interface (i.e., addition of biotin, streptavidin, and antibody), thus allowing for temperature-induced regeneration and disruption of the interface architecture of the electrode surface. Using our immunosensor, we demonstrate sequential amperometric detection of three tumor markers: CA125, CEA, and PSA. Interestingly, greatly amplified signals are achieved by immersing the immunosensor in a solution of horseradish peroxidase (HRP) and antibody-labeled nanoparticles, resulting in a linear range of 0.0064 to 256 U/mL, 1 pg/mL to 100 ng/mL, and 10 pg/mL to 10 ng/mL with a detection limit of 0.007 U/mL, 0.7 pg/mL, and 0.9 pg/mL for CA125, CEA, and PSA, respectively. By alternating temperature, the immunosensor adsorbs and desorbs the biological elements without damage. Our proposed methodology can be expanded to measure other relevant biological species by repeated detection and thus has enormous potential for industrial and clinical applications.

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.

High-purity isolation and recovery of circulating tumor cells using conducting polymer-deposited microfluidic device.

We have developed a conductive nano-roughened microfluidic device and demonstrated its use as an electrically modulated capture and release system for studying rare circulating tumor cells (CTCs). The microchannel surfaces were covalently decorated with epithelial cancer-specific anti-EpCAM antibody by electrochemical deposition of biotin-doped polypyrrole (Ppy), followed by the assembly of streptavidin and biotinylated antibody. Our method utilizes the unique topographical features and excellent electrical activity of Ppy for i) surface-induced preferential recognition and release of CTCs, and ii) selective elimination of non-specifically immobilized white blood cells (WBCs), which are capable of high-purity isolation of CTCs. In addition, the direct incorporation of biotin molecules offers good flexibility, because it allows the modification of channel surfaces with diverse antibodies, in addition to anti-EpCAM, for enhanced detection of multiple types of CTCs. By engineering a series of electrical, chemical, and topographical cues, this simple yet efficient device provides a significant advantage to CTC detection technology as compared with other conventional methods.

Nanoscale label-free bioprobes to detect intracellular proteins in single living cells.

Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells.

Triple-negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti-proliferative effect in cell lines of the basal-like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI-103 combination significantly reduced the level of both phospho-AKT and phospho-ERK in two susceptible BL subtype cell lines, SUM149PT and MDA-MB-468, whereas it had little or no effect on the level of phospho-ERK in two non-susceptible cell lines (HS578T and MDA-MB-231) of mesenchymal stem-like (MSL) TNBC subtype. The gefitinib/PI-103 combination also significantly induced caspase-3/7-mediated PARP cleavage and reduced two anti-apoptotic proteins, XIAP and Bcl-2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl-1) protein was markedly decreased by gefitinib/PI-103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.