Pan W.,Gittlen Cancer Research Foundation
Methods in molecular biology (Clifton, N.J.) | Year: 2010
Aptamers are high-affinity oligonucleotides which can be selected from large random libraries by systematic evolution of ligands by exponential enrichment (SELEX) protocols, with affinities and specificities comparable or better than antibodies. The SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere (with false positives and negatives) with the selection process. Here we present a primer-free protocol using a random DNA library.
Dean S.L.,Pennsylvania State University |
Morrow T.J.,Pennsylvania State University |
Patrick S.,Gittlen Cancer Research Foundation |
Li M.,Pennsylvania State University |
And 3 more authors.
Langmuir | Year: 2013
Combining biological molecules with integrated circuit technology is of considerable interest for next generation sensors and biomedical devices. Current lithographic microfabrication methods, however, were developed for compatibility with silicon technology rather than bioorganic molecules, and consequently it cannot be assumed that biomolecules will remain attached and intact during on-chip processing. Here, we evaluate the effects of three common photoresists (Microposit S1800 series, PMGI SF6, and Megaposit SPR 3012) and two photoresist removers (acetone and 1165 remover) on the ability of surface-immobilized DNA oligonucleotides to selectively recognize their reverse-complementary sequence. Two common DNA immobilization methods were compared: adsorption of 5′-thiolated sequences directly to gold nanowires and covalent attachment of 5′-thiolated sequences to surface amines on silica coated nanowires. We found that acetone had deleterious effects on selective hybridization as compared to 1165 remover, presumably due to incomplete resist removal. Use of the PMGI photoresist, which involves a high temperature bake step, was detrimental to the later performance of nanowire-bound DNA in hybridization assays, especially for DNA attached via thiol adsorption. The other three photoresists did not substantially degrade DNA binding capacity or selectivity for complementary DNA sequences. To determine whether the lithographic steps caused more subtle damage, we also tested oligonucleotides containing a single base mismatch. Finally, a two-step photolithographic process was developed and used in combination with dielectrophoretic nanowire assembly to produce an array of doubly contacted, electrically isolated individual nanowire components on a chip. Postfabrication fluorescence imaging indicated that nanowire-bound DNA was present and able to selectively bind complementary strands. © 2013 American Chemical Society.
Sioss J.A.,Pennsylvania State University |
Bhiladvala R.B.,Pennsylvania State University |
Pan W.,Gittlen Cancer Research Foundation |
Li M.,Pennsylvania State University |
And 7 more authors.
Nanomedicine: Nanotechnology, Biology, and Medicine | Year: 2012
There is widespread interest in circulating tumor cells (CTCs) in blood. Direct detection of CTCs (often < 1/mL) is complicated by a number of factors, but the presence of ~103 to 104 copies of target RNA per CTC, coupled with simple enrichments, can greatly increase detection capability. In this study we used resonance frequency shifts induced by mass-amplifying gold nanoparticles to detect a hybridization sandwich bound to functionalized nanowires. We selected PCA3 RNA as a marker for prostate cancer, optimized antisense binding sites, and defined conditions allowing single nucleotide mismatch discrimination, and used a hybrid resonator integration scheme, which combines elements of top-down fabrication with strengths of bottom-up fabrication, with a view to enable multiplexed sensing. Bound mass calculated from frequency shifts matched mass estimated by counting gold nanoparticles. This represents the first demonstration of use of such nanoresonators, which show promise of both excellent specificity and quantitative sensitivity. From the Clinical Editor: Cancer cell detection from blood is an emerging method for more sensitive screening for malignancies. In this work, RNA detection with nanoresonators is demonstrated to have high specificity and sensitivity, suggesting that such technology may be feasible for laboratory medicine-based cancer detection. © 2012 Elsevier Inc.