Patterned Gold Surface Research Articles

In situ velocity control of gliding microtubules with temperature monitoring by fluorescence excitation on a patterned gold thin film

Microtubule (MT) gliding on a kinesin-coated surface is a promising nanoactuator to manipulate nanomaterials in microfluidic environments. However, controllability of motors with respect to velocity, direction, and lifetime has been challenging for engineering purposes. Here, we used fluorescence excitation to control the MT velocity on a photolithographically patterned gold surface. The excitation wavelength was selected to match that used for the observation of MTs. Since a resistance temperature detector (RTD) was integrated on the assay substrate on which kinesin motors were coated, in situ temperature monitoring was implemented. Compared with the velocity of gliding MTs on the bare glass surface, the velocity increased by 1.8-fold on the gold-coated surface with the increase of temperature of 10.4 °C, which was caused by irradiance of 13.5 W · cm−2. We achieved repetitive velocity control, which was solely caused by the increase of temperature, i.e., irradiation energy. This key technology development enables reversible and localized velocity control of MT gliding, which can be easily integrated in nanosystems driven by kinesin motors.

Abstract 4828: Circulating tumor cells (CTCs) isolated from patients with metastatic breast cancer utilizing a sensitive microfluidic graphene oxide (GO) nanosheet device express markers of cancer stem cells

Abstract Breast cancers are driven by a subset of cells that display stem cell properties. These “cancer stem cells” (CSCs) mediate metastasis and contribute to treatment resistance. Many current CTC isolation technologies are limited in their sensitivity and are particularly inefficient at capturing cells which display an epithelial mesenchymal transition (EMT) phenotype which has been associated with CSCs. We have developed a sensitive microfluidic device using functionalized graphene oxide (GO) nanosheets to identify and isolate CTCs from patients with metastatic breast cancer. Unlike other CTC capture devices with microposts, the effective functionalized surface created by GO allows the device to be two-dimensional. The GO nanosheets were adsorbed onto the patterned gold surface and then chemically functionalized. Blood samples were collected into EDTA and AdnaGen tubes from patients with metastatic breast cancer and analyzed within 24 hours of blood sampling. Isolated CTCs were studied for expression of genes using a multiplex TaqMan-based qRT-PCR method. CTC preparations were checked for white blood cells (WBCs) contamination by examining CD45 expression and verifying that there was no or extremely low levels of CD45 expression in all of the processed samples. The majority of isolated CTCs showed mRNA expression of EMT markers including TGFβ and Vimentin at different levels among tested samples. In addition, markers of breast CSCs including CD44+/CD24- and ALDH1+ were observed in CTCs isolated from patients across the molecular subtypes of breast cancer. Interestingly, HER2+ CTCs were isolated from patients with HER2/neu- primary tumors. This is consistent with recent work from our group that demonstrated selective expression of HER2 in the CSC-like populations of luminal breast cancers occur independent of HER2 gene amplification. These studies demonstrate the feasibility of utilizing GO nanosheet microfluidic devices to isolate and molecularly characterize CTCs from metastatic breast cancer patients. Such liquid biopsies may be of particular value for following CSC populations for patients on therapeutic clinical trials. Citation Format: Shamileh Fouladdel, Hyeun Joong Yoon, Tae Hyun Kim, An Shi, Tahra Luther, Jill Hayden, Shawn G. Clouthier, Yadwinder S. Deol, Monika L. Burness, Daniel F. Hayes, Sunitha Nagrath, Ebrahim Azizi, Max S. Wicha. Circulating tumor cells (CTCs) isolated from patients with metastatic breast cancer utilizing a sensitive microfluidic graphene oxide (GO) nanosheet device express markers of cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4828. doi:10.1158/1538-7445.AM2014-4828

DNA Polymerase as a Molecular Motor and Pump

DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.

Sensitive capture of circulating tumour cells by functionalized graphene oxide nanosheets

The spread of cancer throughout the body is driven by circulating tumour cells (CTCs)1. These cells detach from the primary tumour and move from the blood stream to a new site of subsequent tumour growth. They also carry information about the primary tumour and have the potential to be valuable biomarkers for disease diagnosis and progression, and for the molecular characterization of certain biological properties of the tumour. However, the limited sensitivity and specificity of current methods to measure and study these cells in patient blood samples prevent the realization of their full clinical potential. The use of microfluidic devices is a promising method for isolating CTCs2, 3; however, the devices are reliant on three-dimensional structures, which limit further characterization and expansion of cells on the chip. Here we demonstrate an effective approach to isolate CTCs from blood samples of pancreatic, breast and lung cancer patients, by using functionalised graphene oxide nanosheets on a patterned gold surface. CTCs were captured with high sensitivity at low concentration of target cells (73% ± 32.4 at 3–5 cells/mL blood).

Directed surface attachment of nanomaterials via coiled-coil-driven self-assembly

Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode.

Two-dimensional patterning of bacterial light-harvesting 2 complexes on lipid-modified gold surface

In a photosynthetic membrane, nano-scale patterns of light-harvesting (LH) pigment-protein complexes play an essential role in capturing photons and ensuring efficient excitation energy transfer. LH complexes 1 and 2 have drawn attention as building blocks of a nano-scale photoelectric device. For obtaining a device with efficiency comparable to that of the natural photosynthesis, a method has to be established for forming a two-dimensional assembly of LH complexes around a metal electrode. In this study, LH2 complexes isolated from Rb. sphaeroides were immobilized on a patterned gold surface. Quenching of photo-excitation energy by gold was prevented through the placement of a self-assembled phospholipid monolayer between the LH2 complexes and the gold surface.

Ordered Polymeric Microhole Array Made by Selective Wetting and Applications for Electrochemical Microelectrode Array

In this paper, we report the microelectrode array fabrication using selective wetting/dewetting of polymers on a chemical pattern which is a simple and convenient method capable of creating negative polymeric replicas using polyethylene glycol (PEG) as a clean and nontoxic sacrificial layer. The fabricated hole-patterned polypropylene film on gold demonstrated enhanced electrochemical properties. The chemical pattern is fabricated by microcontact printing using octadecanethiol (ODT) as an ink on gold substrate. When PEG is spin-cast on the chemical pattern, PEG solution selectively dewets the ODT patterned areas and wets the remaining bare gold areas, leading to the formation of arrayed PEG dots. A negative replicas of the PEG dot array is obtained by spin-coating of polypropylene (PP) solution in hexane which preferentially interacts with the hydrophobic ODT region on the patterned gold surface. The arrayed PEG dots are not affected the during PP spin-coating step because of their intrinsic immiscibility. Consequently, the hole-patterned PP film is obtained after PEG removal. The electrochemical signal of the PP film demonstrates the negligible leakage current by high dielectric and self-healing of defects on the chemical pattern by the polymer. This method is applicable to fabrication of microelectrode arrays and possibly can be employed to fabricate a variety of functional polymeric structures, such as photomasks, arrays of biomolecules, cell arrays, and arrays of nanomaterials.

Fluorescence of Two-dimensional Patterned Photosynthetic Proteins onto Gold surface

ABSTRACTPhotosynthetic membrane proteins convert solar light into chemical energy in a significantly high efficiency. Up-to-date reports of the photosynthetic bacterium suggest that such effective light conversion is due to the energy transfer between two light-harvesting (LH) protein complexes that are patterned in two dimensions. In this report, LH complex isolated fromRb. sphaeroideswas immobilized onto a patterned gold surface with self-assembled monolayers (SAMs) and lipid bilayers at two main objectives: (1) micron-scale patterning of LH complex, and (2) prevention of quenching for pattern observation.

Reversible positioning at submicrometre scale of carbon nanotubes mediated by pH-sensitive poly(amino-methacrylate) patterns

The selective adsorption of carbon nanotubes (CNTs) on a pH-reversible PDMAEMA patterned gold surface is presented. In acidic conditions, a selective CNTs adsorption onto the polymer brushes is obtained due to ammonium-π interactions. The reversible behavior was shown by successive treatments in both alkaline and acidic solutions with CNTs.

Have flagella a preferred orientation during early stages of biofilm formation?: AFM study using patterned substrates

Biofilm development involves several stages and flagellar expression of bacteria is considered an important factor in this process. However, its role in the earliest stage of biofilm development is not yet clear. In order to analyse this topic, Pseudomonas fluorescens samples were trapped on a patterned gold surface with sub-microtrenches (ST) so as to hinder their motility, and nanostructured gold with random orientation (SR) was used as control substrate. Atomic force microscopic (AFM) observations were made on untreated samples. Initially, ca. 75% of the flagella on ST and 85% of flagella on SR are oriented towards the neighbouring bacteria. Some of them made contact and surrounded the cells. Subsequently, 2-D raft structures formed on SR inert substrates with lateral curly flagella, while those at the poles of the rafts turned towards the nearest cell group. A few flagella and the formation of 3-D bacterial structures were observed on toxic substrates like copper. Results showed that patterned substrates are suitable tools to detect the orientation of flagella in the earliest stage of biofilm formation on solid opaque surfaces avoiding sample pre-treatment.

Micropatterned Dynamically Adhesive Substrates for Cell Migration

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.

Fabrication of Nanoshell Arrays Using Directed Assembly of Nanospheres

Extraordinary transmission of light has been observed when light is incident on periodic nanostructures patterned metal films, which results from the interaction between the incident photons and the excited surface plasmon polaritons with wavenumbers constrained by the geometry and periodicity of the surface structure. Usually, the surface patterns are fabricated by Focused Ion Beam (FIB) milling, which is expensive and has low throughput. We employ the nanosphere lithography method to fabricate periodic nanoshell arrays on a gold film as an alternative to FIB milling. In this process, polystyrene nanospheres are deposited on glass substrates as a template for subsequent gold deposition. This results in the close-packed hexagonal nanoshell arrays patterned gold surface. The transmission spectra of the patterned surfaces show strong enhanced transmission in the red and IR wavelength regions, and the peak enhanced transmission wavelengths vary with the chemical environments above the surface.

Self-assembly works for superlattices

It is possible to modify the electronic structure of a naturally patterned gold surface in potentially useful ways by allowing layers of silver or copper atoms to self-assemble into arrays of nanostructures.

Template-Directed Adsorption of Block Copolymers on Alkanethiol-Patterned Gold Surfaces

Functionalized alkanethiols have been self-assembled on gold to modify the wetting properties of the surface and promote or hinder the adsorption of block copolymers containing both hydrophobic and hydrophilic blocks. X-ray photoelectron spectroscopy (XPS) studies of spin-coated polyethylene-block-poly(ethylene oxide) (PE-b-PEO) copolymers on 16-mercaptohexadecanoic acid (MHDA)-, octadecanethiol (ODT)-, and 1H,1H,2H,2H-perfluorodecanethiol (PFDT)-covered surfaces have been performed. In the case of an 80 wt % PEO block copolymer, spin-coating on a gold surface precovered with MHDA results in a polymer film thick enough to completely attenuate Au 4f photoelectrons; spin-coating on the more hydrophobic ODT and PFDT monolayers leads to significantly thinner polymer films and incomplete attenuation of the gold photoelectrons. The opposite results are observed when a 20 wt % PEO block copolymer is used. Angle-resolved XPS studies of the 80 wt % PEO block copolymer spin-coated onto an MHDA-covered surface indicate that the PE blocks of the polymer segregate to the near-surface region, oriented away from the hydrophilic carboxylic acid tails of the monolayers; the surface concentration of PE is further enhanced by annealing at 90 degrees C. Microcontact printing and dip-pen nanolithography have been used to pattern gold surfaces with MHDA, and the surfaces have been backfilled with ODT or PFDT, such that the unpatterned regions of the surface are covered with hydrophobic monolayers. In the case of backfilling with PFDT, spin-coating the 80 wt % PEO copolymer onto these patterned surfaces and subsequent annealing results in the block copolymer preferentially adsorbing on the MHDA-covered regions and forming well-defined patterns that mimic the MHDA pattern, as determined by scanning electron microscopy and atomic force microscopy. Significantly worse patterning, characterized by micron-sized polymer droplets, results when the surface is backfilled with ODT instead of PFDT. Using PFDT and MHDA, polymer features having widths as small as 500 nm have been formed. These studies demonstrate a novel method to pattern block copolymers with nanoscale resolution.

Parallel Manipulation of Bifunctional DNA Molecules on Structured Surfaces Using Kinesin‐Driven Microtubules

We have developed a technique to manipulate bifunctional DNA molecules: One end is thiolated to bind to a patterned gold surface and the other end is biotinylated to bind to a microtubule gliding over a kinesin-coated surface. We found that DNA molecules can be stretched and overstretched between the gold pads and the motile microtubules, and that they can form dynamic networks. This serves as a proof-of-principle that biological machineries can be used in vitro to accomplish the parallel formation of structured DNA templates that will have applications in biophysics and nanoelectronics.

Submerged Microcontact Printing (SμCP): An Unconventional Printing Technique of Thiols Using High Aspect Ratio, Elastomeric Stamps

A technique for microcontact printing of thiols in liquid media is presented. Elastomeric poly(dimethyl siloxane) stamps are used to pattern gold surfaces with thiol-based self-assembled monolayers. The liquid (water in this case) has been used as an incompressible support and, advantageously, also acts as a medium in which alkylthiol ink molecules are poorly miscible. Consequently, we have been able to produce patterned thiol monolayers using stamps with aspect ratios unsuitable for conventional microcontact printing (i.e., 15:1) and present evidence to suggest that it is possible to use stamps with aspect ratios of up to 100:1.

Micro‐Imprinting of Oligonucleotides and Oligonucleotide Gradients on Gold Surfaces: A New Approach Based on the Combination of Scanning Electrochemical Microscopy and Surface Plasmon Resonance Imaging (SECM/ SPR‐i)

AbstractA new strategy for the formation of DNA patterns on thin gold surfaces together with DNA hybridization is described. The formation and characterization of the oligonucleotide array is based on a combination of an electrochemical and an optical technique: scanning electrochemical microscopy and surface plasmon resonance imaging (SECM/SPR‐i). The DNA array is formed through electropolymerization of a mixture of pyrrole and pyrrole bearing covalently linked oligonucleotides probes using SECM. SPR‐i as well as fluorescence imaging are used to quantify DNA hybridization on these multi‐parametric interfaces. The capability of scanning electrochemical microscopy (SECM) to pattern gold surfaces with density gradients of oligonucleotides attached to the gold surfaces is further demonstrated. These density gradients result from the progressive increase in the movement of the x‐axis while applying continuous potential pulses to the gold surface. Surface plasmon resonance imaging is used to detect the deposition step, while fluorescence imaging is employed to visualize the deposited oligonucleotide density gradient.

Field emission from single-walled carbon nanotubes aligned on a gold plate using a self-assembly monolayer

Field emission from single-walled carbon nanotubes (SWNTs) aligned on a patterned gold surface is reported. The SWNT emitters were prepared at room temperature by a self-assembly monolayer technique. SWNTs were cut into sub-micron lengths by sonication in an acidic solution. Cut SWNTs were attached to the gold surface by the reaction between the thiol groups and the gold surface. The field-emission measurements showed that the turn-on field was 4.8 V/μm at an emission current density of 10 μA/cm2. The current density was 0.5 mA/cm2 at 6.6 V/μm. This approach provides a novel route for fabricating CNT-based field-emission displays.

Patterned monolayer/polymer films for analysis of dilute or salt-contaminated protein samples by MALDI-MS.

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-microm diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance FT-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.