Fabrication of gold nanoparticle modified ITO substrate to detect beta-amyloid using surface-enhanced Raman scattering.

Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the deposition of beta-amyloid (Abeta) peptide and the formation of neurofibrillary tangles in neurons. The Abeta peptide is a key molecule in the pathogenesis of Alzheimer's disease and an important marker for early diagnosis. Surface-enhanced Raman scattering (SERS) has recently been attracting keen interest in various fields such as for biosensors or immunoassays. In this study, gold nanoparticles (Au NPs) were electrochemically deposited on an indium tin oxide (ITO) substrate at different heights. Abeta antibodies were immobilized on the Au-NP-coated ITO substrate, after which the interactions between the antigen and the antibody were determined via SERS spectroscopy. The SERS responses were strongest at the Au NP array height of 91 nm, with a good linear relationship that corresponded to the change in the concentration of the antigen. This Au-NP-array-mediated SERS can be applied with a highly sensitive immunodetection biosensor.

Detection of the effect of environmental toxicants on the synthetic peptide modified neural cell chip.

A cell based chip was designed to differentiate and to detect the effects of environmental chemicals on the neurite outgrowth in PC12 cell. To fabricate platform of cell chip, gold surfaces were modified by RGD based synthetic oligopeptide. Nanoscale controlled self-assembled peptide layer was investigated by Atomic Force Microscopy (AFM). On the fabricated cell chip, PC12 cell was immobilized and the differentiation of neurite outgrowth in PC12 cells was done by neurite growth factor (NGF). Differentiation of PC12 cell was confirmed by immunofluorescence study. Further the differentiation and the length of neurite was confirmed by confocal microscopy study. Voltammetry behavior of the neurite induced PC12 and the electrochemical behavior of the environmental toxicants effect on the neurite outgrowth was measured by cyclic voltammetry (CV). Self-assembled layer mediated cell immobilization technique and voltammetric signal analysis system can be applied to construct the neural cell chip for the detection of large number of environmental toxins and various neurotoxicants.

Surface-enhanced Raman scattering of dopamine on self-assembled gold nanoparticles.

Dopamine, a potent neurotransmitter in the brain, influences a variety of motivated behaviors and plays a major role in Parkinson's disease. In this study, the Raman signal of dopamine was detected on a fabricated nanoparticle-immobilized glass surface by surface-enhanced raman spectroscopy (SERS). Amine-modified glass was prepared by the self-assembly of amine-terminated silane on substrate, followed by the deposition of gold nanoparticles. The gold nanoparticles deposited on the glass surface were functionalized by anti-dopamine or dopamine. The antigen-dopamine was captured by antibody-assembled gold substrate and detected by SERS. The optical properties and morpology of the glass substrate with immobilized gold nanoparticles were analyzed by scanning electron microscopy and UV-VIS absorption spectroscopy. The Raman spectrum of dopamine displayed broad bands at 1267, 1331, 1158, 1478, 1578 and 1584 cm(-1). The strongest peaks in the spectra (at 1267 and 1478 cm(-1)) were identified as phenolic carbon-oxygen and phenyl C=C stretches, respectively. A working curve of the SERS signal constructed from cathecol ring vibration versus antigen-dopamine concentration was obtained at 1478 cm(-1), and the non-optimized detection limit for anti-dopamine surface antigen was as low as 1 ng/ml. These results suggest that SERS-based immunosensor can be a promising tool for the detection and screening of neurotransmitters.

Direct immobilization of cupredoxin azurin modified by site-directed mutagenesis on gold surface.

For making efficient bioelectronic device, we have developed novel immobilization method of cupredoxin azurin modified on gold (Au) surface. A recombinant protein with cysteine residue by using site-directed mutagenesis was designed and then directly immobilized on Au surface without any chemical linker. The immobilization of the functionalized protein is confirmed by surface plasmon resonance (SPR) and its surface morphology is analyzed by scanning tunneling microscopy (STM). The immobilization efficiency has been increased about 75.6%, as compared to that of wild-type azurin. The electrochemical property of the fabricated thin film was investigated by the cyclic voltammetry (CV). As a result, cysteine-modified azurin can be used for making high-quality protein film, and applied to the fabrication of nano-scale bioelectronics.

The immobilization of animal cells using the cysteine-modified RGD oligopeptide.

RGD peptide sequence is an effective cell recognition motif and used to enhance the cell adhesion on desired solid material for cell immobilization. We have synthesized CRGD, CRGD-multiple-armed peptide (MAP), RGD-MAP-C and evaluated their comparative efficacy for cell immobilization. Each peptide was assembled on gold surface and investigated by the atomic force microscopy (AFM) technique in the contact mode. The viability of immobilized animal cells was examined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our results showed that RGD-MAP-C in comparison to others was the most effective proliferation of cells on the gold surface. The goal of this present work is integration to the nano-pattern cell chip bioplatform for biomedical assays or provide valuable insights into cell biology and design of biomaterials. This RGD-MAP-C can be applicable to the nano-pattern cell chip platform.

Fabrication of recombinant azurin self-assembled layer for the application of bioelectronic device.

A novel immobilization method of cupredoxin azurin on a gold surface was developed without a chemical linker using recombinant technique. A recombinant protein with cysteine residue by site-directed mutagenesis (SDM) was designed and then directly self-assembled on Au surface. The layer of the functionalized protein immobilized is confirmed by surface plasmon resonance (SPR) and its surface morphology is analyzed by scanning tunneling microscopy (STM). The immobilization efficiency has been increased about 73.2%, as compared to that of wild type azurin. The electrochemical property of the fabricated thin layer was investigated by the cyclic voltammetry (CV). As a result, cysteine-substituted azurin can be used for making high quality protein film, and applied to the fabrication of nano-scale bioelectronics.

In situ label-free quantification of human pluripotent stem cells with electrochemical potential.

Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein, we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs, their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs, which can assess the risk of teratoma formation efficiently and economically.

Large-Scale Nanoelectrode Arrays to Monitor the Dopaminergic Differentiation of Human Neural Stem Cells.

A novel cell-based biosensing platform is developed using a combination of sequential laser interference lithography and electrochemical deposition methods. This enables the sensitive discrimination of dopaminergic cells from other types of neural cells in a completely nondestructive manner. This platform and detection strategy may become an effective noninvasive in situ monitoring tool that can be used to determine stem cell fate for various regenerative applications.

Synthesis of metal nanoparticles inside living human cells based on the intracellular formation process.

Intracellular and extracellular formation of Au and Ag NPs with different sizes and shapes using human cells has been developed as green method, which does not require the use of any reducing agents. Also, the cell lysis is used for production of different metal NPs. Our results demonstrate that treatment of human cells with various metal ions cause cell fixation.

Cell chip with a thiolated chitosan self-assembled monolayer to detect the effects of anticancer drugs on breast normal and cancer cells.

Cell-based chips are an effective in vitro analysis tool; however, the sensitivity of the cell chip to biomaterials is high, which is crucial for immobilizing cells on the electrode surface without conductivity. In this study, we report on a cell chip with a thiolated chitosan monolayer that was easy to fabricate, highly adhesive to cells, and enhanced electrochemical signals. Thiolated chitosan containing thiol groups was synthesized and self-assembled on a gold electrode to immobilize cells, and showed superior electrochemical performance to that of poly-l-lysine and collagen. Cyclic voltammetry (CV) was performed to distinguish the redox characteristics of normal (HMEC) and breast cancer cells (MCF-7); then, two anticancer drugs (doxorubicin and cyclophosphamide) were added to the cell cultures to analyze their effects on the redox environment of normal and cancer cells derived from the same origin. As a result, the CV cathode peaks decreased differently with respect to the cell line (normal and cancer) and anticancer drug, which was validated by a conventional MTT viability assay. Hence, the proposed cell chip with a thiolated chitosan modified layer could be used in various fields, including discriminating normal from cancer cells, to evaluating the efficiency of newly developed drugs, and to assessing cytotoxicity of various chemicals.

In situ electrochemical detection of embryonic stem cell differentiation.

Stem cell sensors have emerged as a promising technique to electrochemically monitor the functional status and viability of stem cells. However, efficient electrochemical analysis techniques are required for the development of effective electrochemical stem cell sensors. In the current study, we report a newly developed electrochemical cyclic voltammetry (CV) system to determine the status of mouse embryonic stem (ES) cells. 1-Naphthly phosphate (1-NP), which was dephosphorylated by alkaline phosphatase into a 1-naphthol on an undifferentiated mouse ES cell, was used as a substrate to electrochemically monitor the differentiation status of mouse ES cells. The peak current in the cyclic voltammetry of 1-NP increased linearly with the concentration of pure 1-NP (R(2)=0.9623). On the other hand, the peak current in the electrochemical responses of 1-NP decreased as the number of undifferentiated ES cells increased. The increased dephosphorylation of 1-NP to 1-naphthol made a decreased electrochemical signal. Non-toxicity of 1-NP was confirmed. In conclusion, the proposed electrochemical analysis system can be applied to an electrical stem cell chip for diagnosis, drug detection and on-site monitoring.

Electrochemical cell chip to detect environmental toxicants based on cell cycle arrest technique.

A cell-based chip was recently developed and shown to be an effective in vitro tool for analyzing effect of environmental toxin on target cells. However, common cell chips are inappropriate for the detection of multiple environmental toxins. Here, we fabricated a neural cell chip to detect different cellular responses induced by BPA (bisphenol-A) and PCB (poly chlorinated biphenyl). This approach was based on an electrochemical method using a cell cycle-arrest technique. Neural cells were synchronized at the synthesis phase by treatment with thymidine, which results in a sharp reduction peak when compared to unsynchronized cells. The fabricated chip containing 50% G1/S and 50% G2/M phase cells was used to determine the effects of environmental toxins on neural cancer cells. At the end, the cell-chips could be used to assess both BPA and PCB toxicity that the cells were completely synchronized at the G1/S and G2/M phase. The proposed neural cell chip can be a useful tool for biosensors to evaluate easily and sensitively multiple effects of environmental toxicants on target cells.

Analysis of effect of nanoporous alumina substrate coated with polypyrrole nanowire on cell morphology based on AFM topography.

In this study, in situ electrochemical synthesis of polypyrrole nanowires with nanoporous alumina template was described. The formation of highly ordered porous alumina substrate was demonstrated with Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). In addition, Fourier transform infrared analysis confirmed that polypyrrole (PP) nanowires were synthesized by direct electrochemical oxidation of pyrrole. HeLa cancer cells and HMCF normal cells were immobilized on the polypyrrole nanowires/nanoporous alumina substrates to determine the effects of the substrate on the cell morphology, adhesion and proliferation as well as the biocompatibility of the substrate. Cell adhesion and proliferation were characterized using a standard MTT assay. The effects of the polypyrrole nanowires/nanoporous alumina substrate on the cell morphology were studied by AFM. The nanoporous alumina coated with polypyrrole nanowires was found to exhibit better cell adhesion and proliferation than polystyrene petridish, aluminum foil, 1st anodized and uncoated 2nd anodized alumina substrate. This study showed the potential of the polypyrrole nanowires/nanoporous alumina substrate as biocompatibility electroactive polymer substrate for both healthy and cancer cell cultures applications.

Effects of nanopatterned RGD peptide layer on electrochemical detection of neural cell chip.

The cell-based chip is becoming a popular tool for monitoring living cell viability under various conditions. In this study, several biomaterials, such as synthetic Cys-(Arg-Gly-Asp)(4) (C(RGD)(4)), Arg-Gly-Asp-Multi Armed-Cys (RGD-MAP-C) peptide, and poly-L-lysine (PLL) nano-dots were fabricated on the gold surface of a neural cell chip. The material-dependent effects both on electrochemical signal detection in neural cells and on cellular adhesion were analyzed. The nano-dot structures were fabricated through a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). PC12 cells were allowed to attach on several peptide nanopatterned surfaces, and electrochemical tools were applied to neural cells attached on the chip surface. The RGD-MAP-C peptide nanopatterned surface provided the strongest voltammetric signals when the cell was exposed to cyclic voltammetry (CV) and differential pulse voltammetry (DPV) after 48 h of incubation, which may largely be due to an enhanced affinity between cells and the Au surface. Chemical toxicity assessments were conducted in the fabricated cell chip, and they showed negative correlations between neural cell viability and the concentration of chemicals. In conclusion, a nanopatterned RGD-MAP-C layer improved cell-binding affinity to Au substrates and showed sufficient sensitivity for electrochemical detection of cell viability.

Electrical detection of beta-amyloid (1-40) using scanning tunneling microscopy.

Numerous studies have shown that the presence of beta-amyloid (1-40) in cerebrospinal fluid can be used as a potential biomarker for Alzheimer's disease. Identifying biomarkers for Alzheimer's disease is highly important because these biomarkers could be used to establish the diagnosis before the disease reaches clinical severity. In this study, a vertically configured electrical detection system associated with scanning tunneling microscopy (STM) was used to characterize antigen-antibody binding interactions. The proposed technique can be easily utilized to construct a multiple measurement system in a protein chip. The immunocomplexes used in the model protein comprise beta-amyloid (1-40), corresponding antibody fragments, and gold nanoparticle-antibody conjugates. The electrical tunneling current between the STM tip and these complexes exhibited a peak-like pulse, where the frequency of these pulses was dependent on the surface density of bound complexes. Hence, a quantitative measurement of beta-amyloid concentration from a periodogram analysis of peak frequency was successfully achieved at concentrations as low as 1fg/mL.

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry.

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.