Utilization of Aptamers as Affinity Probes in Polymeric Microdevices for Disease Management and the Production of a Recombinant Membrane Protein

Aptamers have emerged as potential affinity agents that rival or complement antibodies in developing diagnostic assays for disease detection. We have demonstrated the use of PMMA microfluidic devices for conducting rapid affinity microchip CGE of a target protein using aptamers as the affinity probes with an electrophoresis development time of \u3c2 min. Migration times were reproducible for thrombin complexes and free aptamers in CGE buffer with device-to-device RSD variations below 10%. By removing salts from plasma and adding an unlabeled random sequence oligonucleotide to the plasma, thrombin was detected in plasma. This method can be easily adapted to high-throughput parallel screening of plasma samples in multi-channel polymeric microdevices. The expression of rEpCAM in bacteria and mammalian cells was investigated with results showing successful expression in the mammalian systems, while protein expression in the bacteria was inhibited. Higher and prolonged expression levels were obtained in Cos 7 cells as compared to BHK and Hep2 cells due to episomal expression in Cos 7 cells. By combining a one-step IMAC procedure with electrophoresis and electro elution a more selective approach to obtaining electrophoretically pure rEpCAM was achieved. We developed dual-aptamer sandwich assays for thrombin and PDGF-BB targets on PMMA substrate using UV modification with a one-step EDC/NHS coupling reaction. Results indicate that the use of high EDC concentrations lead to faster immobilization and increased coverage density with a shorter sandwich assay development time. The assays were sensitive for both proteins and showed linearity in fluorescence response to changing protein concentration. Single molecule detection methods provide high sensitivity techniques in which aptamers can find use. We have demonstrated the use of two aptamers in a FRET-based assay for the detection of low levels of thrombin. Sensitivity and reliability of the aptamer smFRET assay depends on complex formation and stability. Due to the dissociation constants of the aptamers, single molecule events detected were below the calculated value for a 6 pM concentration of thrombin. Future work will involve adjusting the single molecule system by the use of pinholes to enable analysis of higher sample concentrations

Screening of Compost for PAHs and Pesticides Using Static Subcritical Water Extraction

Static subcritical water extraction (SubWE) along with solid phase extraction (SPE) was used for the analysis of PAHs and pesticides in municipal solid waste compost. Yields obtained for PAHs in certified reference sediment (CRM 104) were acceptable. The extraction method was simple, rapid, used small sample sizes, and no sample drying was required. Analysis of samples was performed by GC/MS and HPLC. Recovery of spiked pesticides was greatest at 110°C for 20 min extraction time. The optimum extraction for PAH analysis was achieved at150°C for 20 min. Addition of C-18 resin as an “alternate sorbent” upon cooling increased recovery of PAHs but not of pesticides, however, it increased the stability of atrazine and propazine at higher temperatures. Analysis of three municipal compost samples from the Dayton, OH (USA) area showed no pesticides above the detection limit, however, PAH totals for 11 PAHs were 15.97, 14.42, and 20.79μgg−1. The totals of six of the seven carcinogenic PAHs, for which remediation goals in the United States is 4.6μgg−1, were determined to be 9.89, 6.77, and 13.06μgg−1 dry weight. The highest PAH totals were obtained from compost containing sewage sludge

Screening of Compost for PAHs and Pesticides Using Static Subcritical Water Extraction

Static subcritical water extraction (SubWE) along with solid phase extraction (SPE) was used for the analysis of PAHs and pesticides in municipal solid waste compost. Yields obtained for PAHs in certified reference sediment (CRM 104) were acceptable. The extraction method was simple, rapid, used small sample sizes, and no sample drying was required. Analysis of samples was performed by GC/MS and HPLC. Recovery of spiked pesticides was greatest at 110°C for 20 min extraction time. The optimum extraction for PAH analysis was achieved at150°C for 20 min. Addition of C-18 resin as an “alternate sorbent” upon cooling increased recovery of PAHs but not of pesticides, however, it increased the stability of atrazine and propazine at higher temperatures. Analysis of three municipal compost samples from the Dayton, OH (USA) area showed no pesticides above the detection limit, however, PAH totals for 11 PAHs were 15.97, 14.42, and 20.79μgg−1. The totals of six of the seven carcinogenic PAHs, for which remediation goals in the United States is 4.6μgg−1, were determined to be 9.89, 6.77, and 13.06μgg−1 dry weight. The highest PAH totals were obtained from compost containing sewage sludge

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. © 2007 Springer-Verlag

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-(CH 2) 6-OP(O) 2O-(CH 2CH 2O) 6-TTTTT-aptamer exhibited a 4-fold increase in binding capacity versus SAMs made using HS-(CH 2) 6-TTTTT-aptamer. Furthermore, SAMs made using HS-(CH 2) 6-OP(O) 2O-(CH 2CH 2O) 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-(CH 2) 6-OP(O) 2O-(CH 2CH 2O) 6-TTTTT-RANDOM) showed little binding of thrombin. Irrespective of the aptamer-linker system, use of HS-(CH 2) 11(OCH 2CH 2) 3OH, referred to as EG 3, as a coadsorbent enhanced binding of thrombin by approximately 2.5-fold compared to that of HS-(CH 2) 6-OH (mercaptohexanol, MCH). © 2006 American Chemical Society

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 (T0, T 5, T10, T20), the surface density of the aptamer decreased linearly from approximately 25 to 12 pmol·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 T5, 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. © 2008 American Chemical Society

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 nonequilibriurn 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 \u3c10% 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. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim