Nanostructure shape effects on response of plasmonic aptamer sensors.

A localized surface plasmon resonance (LSPR) sensor surface was fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor showed a response of 6-nm λ(max) shift for protein binding with the detection limit of at least 10 pM, indicating one of the highest sensitivities achieved for thrombin detection by optical extinction LSPR. We also tested the LSPR sensor fabricated using gold bipyramid, which showed higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. XPS analysis showed that this is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The low surface density of the aptamers on the gold bipyramid surface may be due to the effect of shape of the nanostructure on the kinetics of aptamer monolayer formation. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR, demonstrated here for protein binding. The generality of aptamer sensors for protein detection using gold nanorod and gold nanobipyramid substrates is anticipated to have a large impact in the important development of sensors toward biomarkers, environmental toxins, and warfare agents.

Molecular imprinting in monolayer surfaces.

A comprehensive report on molecularly imprinted monolayers (MIMs) is presented, but does not include bulk-polymer thin film coatings on surfaces, inorganic surface imprinting, polymer grafting and layer-by-layer methods. Due to difficulties in imprinting large molecules and obtaining fast binding responses with traditional network polymer materials, MIMs have been developed with the aim of enhancing mass-transfer of analytes in imprinted materials. Three approaches to MIM fabrication have been developed with respect to the formation of the pre-organized template-matrix complex. In the first approach, the molecular binding sites are formed in a monolayer on a glass or gold surface. The second approach uses a template-macromolecule complex to form binding sites in the solution phase that are immobilized onto a surface; and the third approach transfers an imprinted Langmuir film onto a gold surface. Mass transfer in these MIMs in most cases is on the order of minutes, and both small and large molecules (proteins) have been imprinted.

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits.

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

Highly efficient capture and enumeration of low abundance prostate cancer cells using prostate-specific membrane antigen aptamers immobilized to a polymeric microfluidic device.

Prostate tumor cells over-express a prostate-specific membrane antigen (PSMA) that can be used as a marker to select these cells from highly heterogeneous clinical samples, even when found in low abundance. Antibodies and aptamers have been developed that specifically bind to PSMA. In this study, anti-PSMA aptamers were immobilized onto the surface of a capture bed poised within a PMMA, microchip, which was fabricated into a high-throughput micro-sampling unit (HTMSU) used for the selective isolation of rare circulating prostate tumor cells resident in a peripheral blood matrix. The HTMSU capture bed consisted of 51 ultra-high-aspect ratio parallel curvilinear channels with a width similar to the prostate cancer cell dimensions. The surface density of the PSMA-specific aptamers on an ultraviolet-modified PMMA microfluidic capture bed surface was determined to be 8.4 x 10(12) molecules/cm(2). Using a linear velocity for optimal cell capture in the aptamer-tethered HTMSU (2.5 mm/s), a recovery of 90% of LNCaP cells (prostate cancer cell line; used as a model in this example) was found. Due to the low abundance of these cells, the input volume required was 1 mL and this could be processed in approximately 29 min using an optimized linear flow rate of 2.5 mm/s. Captured cells were subsequently released intact from the affinity surface using 0.25% w/w trypsin followed by counting individual cells using a contact conductivity sensor integrated into the HTMSU that provided high detection and sampling efficiency (approximately 100%) and did not require staining of the cells for enumeration.

Effect of linker structure on surface density of aptamer monolayers and their corresponding protein binding efficiency.

A systematic study is reported on the effect of linker size and its chemical composition toward ligand binding to a surface-immobilized aptamer, measured using surface plasmon resonance. The results, using thrombin as the model system, showed that as the number of thymidine (T) units in the linker increases from 0 to 20 in four separate increments (T(0), T(5), T(10), T(20)), the surface density of the aptamer decreased linearly from approximately 25 to 12 pmol x cm(-2). The decrease in aptamer surface density occurred due to the increased size of the linker molecules. In addition, thrombin binding capacity was shown to increase as the linker length increased from 0 to 5 thymidine nucleotides and then decreased as the number of thymidine residues increased to 20 due to a balance between two different effects. The initial increase was due to increased access of thrombin to the aptamer as the aptamer was moved away from the surface. For linkers greater in length than T(5), the overall decrease in binding capacity was primarily due to a decrease in the surface density. Incorporation of a hexa(ethylene glycol) moiety into the linker did not affect the surface density but increased the amount of thrombin bound. In addition, the attachment of the linker at the 3'- versus the 5'-end of the aptamer resulted in increased aptamer surface density. However, monolayers formed with equal surface densities showed similar amounts of thrombin binding irrespective of the point of attachment.

Poly(methyl methacrylate) microchip affinity capillary gel electrophoresis of aptamer-protein complexes for the analysis of thrombin in plasma.

Thrombin generation in blood serves as an important marker for various hemostasis-related diseases and conditions. Analytical techniques currently utilized for determining the thrombin potential of patients rely primarily on the enzymatic activity of thrombin. Microfluidic-based ACE using fluorescently labeled aptamers as affinity probes could provide a simple and efficient technique for the real-time analysis of thrombin levels in plasma. In this study, aptamers were used for the analysis of thrombin by affinity microchip CGE. The CGE used a poly(methyl methacrylate) (PMMA) microfluidic device for the sorting of the affinity complexes with a linear polyacrylamide (LPA) serving as the sieving matrix. Due to the fact that the assay was run under nonequilibrium electrophoresis conditions, the presence of the sieving gel was found to stabilize the affinity complex, providing improved electrophoretic performance compared to free-solution electrophoresis. Two fluorescently labeled aptamer affinity probes, HD1 and HD22, which bind to exosites I and II, respectively, of thrombin were investigated. With an electric field strength of 300 V/cm, two well-resolved peaks corresponding to free aptamer and the thrombin-aptamer complex were obtained in less than 1 min of separation time with a run-to-run and chip-to-chip reproducibility (RSD) of migration times <10% using both aptamers. HD22 affinity assays of thrombin produced baseline-resolved peaks with favorable efficiency due to its higher binding affinity, whereas HD1 assays showed poorer resolution of the free aptamer and complex peaks. HD22 was used in determining the level of thrombin in human plasma. Assays were performed directly on plasma that was diluted to 10% v/v. Thrombin was successfully analyzed by microchip CGE at a concentration level of 543.5 nM for the human plasma sample.

Surface immobilization methods for aptamer diagnostic applications.

In this review we examine various methods for the immobilization of aptamers onto different substrates that can be utilized in a diverse array of analytical formats. In most cases, covalent linking to surfaces is preferred over physisorption, which is reflected in the bulk of the reports covered within this review. Conjugation of aptamers with appropriate linkers directly to gold films or particles is discussed first, followed by methods for conjugating aptamers to functionally modified surfaces. In many aptamer-based applications, silicates and silicon oxide surfaces provide an advantage over metallic substrates, and generally require surface modification prior to covalent attachment of the aptamers. Chemical protocols for covalent attachment of aptamers to functionalized surfaces are summarized in the review, showing common pathways employed for aptamer immobilization on different surfaces. Biocoatings, such as avidin or one of its derivatives, have been shown to be highly successful for immobilizing biotin-tethered aptamers on various surfaces (e.g., gold, silicates, polymers). There are also a few examples reported of aptamer immobilization on other novel substrates, such as quantum dots, carbon nanotubes, and carbohydrates. This review covers the literature on aptamer immobilization up to March 2007, including comparison of different linkers of varying size and chemical structure, 3' versus 5' attachment, and regeneration methods of aptamers on surfaces.

Designing highly specific biosensing surfaces using aptamer monolayers on gold.

To build highly specific surfaces using aptamer affinity reagents, the effects of linker and coadsorbents were investigated for maximizing target binding and specificity for aptamer-based self-assembled monolayers (SAMs) supported on gold. An aptamer that binds the protein thrombin was utilized as a model system to compare different mixed monolayer systems toward maximizing binding and selectivity to the immobilized aptamer. Important factors used to optimize binding characteristics of thrombin to the aptamer-based monolayer films include changes in design elements of the linker and different coadsorbent thiols. Binding events measured by surface plasmon resonance (SPR) and ellipsometry showed that the binding performance of the aptamer SAMs depends principally on the linker and to a lesser extent on the coadsorbent. SAMs formed with HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer exhibited a 4-fold increase in binding capacity versus SAMs made using HS-(CH2)6-TTTTT-aptamer. Furthermore, SAMs made using HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer showed nearly complete specificity for thrombin versus bovine serum albumin (BSA, less than 2% bound), while a SAM incorporating a random DNA fragment (HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-RANDOM) showed little binding of thrombin. Irrespective of the aptamer-linker system, use of HS-(CH2)11(OCH2CH2)3OH, referred to as EG(3), as a coadsorbent enhanced binding of thrombin by approximately 2.5-fold compared to that of HS-(CH2)6-OH (mercaptohexanol, MCH).

Reversible protein adsorption and bioadhesion on monolayers terminated with mixtures of oligo(ethylene glycol) and methyl groups.

Surface-grafted, environmentally responsive polymers have shown great promise for controlling adsorption and desorption of macromolecules and cells on solid surfaces. In the paper, we demonstrate that certain mixed self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG) and methyl-terminated alkanethiolates on gold form surfaces with switchable hydrophobicity and tendency for protein adsorption and cellular attachment. At temperatures above 32 degrees C, SAMs with a surface density of approximately 50% OEG adsorbed significant amounts of pyruvate kinase and lysozyme, whereas below this temperature, these same SAMs were resistant to the adsorption of these proteins. Furthermore, protein layers adsorbed to these SAMs above 32 degrees C were removed upon rinsing with water below this temperature. Similar results were seen for attachment and release of the marine bacterium, Cobetia marina. The change from nonresistance to adsorptive state of the SAMs was concomitant with a change in advancing water contact angle. Vibrational sum frequency generation spectroscopy suggests that the temperature-induced changes coincide with a disorder-to-partial order transition of the hydrated methylene chains of the OEG moieties within the SAMs. Mixed OEG-methyl SAMs represent both a convenient means of controlling macromolecular and cellular adsorption within the laboratory and a useful tool for relating adsorption properties to molecular structures within the SAMs.