Systematic Investigation of Optimal Aptamer Immobilization for Protein−Microarray Applications

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to a wide range of target molecules with high affinity and specificity. As nucleic acids, aptamers can undergo denaturation, but the process is reversible. As a result of this stability and the possibility of automated selection of aptamers, these oligonucleotides are highly promising capture molecules in microarray formats. In this study, his-tagged proteins and an aptamer directed against the his-tag were chosen as a model system. Different factors affect the activity of aptamers immobilized on a solid support like a microarray surface. The orientation of the immobilized aptamer plays an important role in correct aptamer folding and, thus, in effective binding of the corresponding target. Other important parameters identified in this work are the microarrays’ surface charge as well as the length of the spacer between aptamer and solid support. These parameters were investigated systematically, resulting in the development of an aptamer-based microarray for detection of his-tagged proteins. The general applicability of the developed immobilization strategy was demonstrated by utilization of three different aptamers.

Aptamer-Based Au Nanoparticles-Enhanced Surface Plasmon Resonance Detection of Small Molecules

Small molecules are difficult to detect by conventional SPR technique directly because the changes in the refractive index resulting from the binding processes of small biomolecules are often small. In order to extend the application of SPR biosensor in detecting a small molecule, we combine the advantage of aptamer technique with the amplifying effect of Au nanoparticles to design a sensitive SPR sensor for detecting small molecules. The principle of this sensor is based on surface inhibition detection. The aptamer is first immobilized on SPR gold film with its ss-DNA structure. The aptamer possessing this structure can be hybridized with Au nanoparticles-tagged complementary ss-DNA and result in a large change of SPR signal. However, the aptamer will change its structure from ss-DNA to tertiary structure after adenosine is added to the SPR cell. The aptamer possessing tertiary structure could not hybridize with Au nanoparticles-tagged complementary ss-DNA. Thus, the change of SPR signal resulted in the hybridization reaction between aptamer and Au nanoparticles-tagged complementary ss-DNA will decrease with the increase of the number of aptamers possessing tertiary structure, which is proportional to the concentration of the small molecule. Based on this principle, we choose a simple system (antiadenosine aptamer/adenosine) to detect the sensing ability of this SPR biosensor for a small molecule. The experimental results confirm that the SPR sensor we developed possesses a good sensitivity and a high selectivity for adenosine. The detection range for adenosine is from 1 × 10⁻⁹ to 1 × 10⁻⁶ M. More significantly, it is fairly easy to generalize this strategy to detect a spectrum of small molecules by SPR spectroscopy using different aptamers. Therefore, it is expected that this method may offer a new direction in designing high-performance SPR biosensors for sensitive and selective detection of a wide spectrum of small molecules.

http://pubs.acs.org/cgi-bin/abstract.cgi/ancham/asap/abs/ac801281c.html

Latent Fingerprint Chemical Imaging by Mass Spectrometry

Latent fingerprints (LFPs) potentially contain more forensic information than the simple identification of the subject; they may contain evidence of contacts with explosives or substances of abuse. Chemical information can also be useful in resolving overlapping LFPs from different individuals. We used desorption electrospray ionization mass spectrometry in an imaging mode to record compound-specific chemical fingerprints.

http://www.sciencemag.org/cgi/reprint/321/5890/805.pdf

Nanotechnology and aptamers: applications in drug delivery

Nucleic acid ligands, also known as aptamers, are a class of macromolecules that are being used in several novel nanobiomedical applications. Aptamers are characterized by high affinity and specificity for their target, a versatile selection process, ease of chemical synthesis and a small physical size, which collectively make them attractive molecules for targeting diseases or as therapeutics. These properties will enable aptamers to facilitate innovative new nanotechnologies with applications in medicine. In this review, we will highlight recent developments in using aptamers in nanotechnology solutions for treating and diagnosing disease.

Aptamer-based biosensors

Nucleic-acid aptamers have attracted intense interest and found wide applications in a range of areas. In this review, we summarize recent advances in the development of aptamer-based biosensors and bioassay methods, most of which have employed electrochemical, optical and mass-sensitive analytical techniques. Aptamers exhibit many advantages as recognition elements in biosensing when compared to traditional antibodies. They are small in size, chemically stable and cost effective. More importantly, aptamers offer remarkable flexibility and convenience in the design of their structures, which has led to novel biosensors that have exhibited high sensitivity and selectivity. Recently, the combination of aptamers with novel nanomaterials has significantly improved the performance of aptamer-based sensors, which we also review in this article. In view of the unprecedented advantages brought by aptamers, we expect aptamer-based biosensors to find broad applications in biomedical diagnostics, environmental monitoring and homeland security.

Keywords: Aptamer; Biosensor; Nanomaterial; Nanotechnology

Aptamers as recognition elements for label-free analytical devices

In spite of the impressive advances in aptasensing, the search for label-free devices for the detection of the aptamer-ligand interaction is still a challenge. This review highlights the advantages and the limitations of using label-free detection strategies and summarizes the state of the art in this field.

Nanostructures as analytical tools in bioassays

We critically evaluate the usefulness of different nanostructures described as labels, nanoscaffolds or separation media in immunoassays and nucleic-acid hybridization assays. Many of the great number of publications describe only theoretical aspects of using these nanostructures or nanoparticles, but do not verify their applicability in the presence of potential interferents that can be present in the sample matrix. We attempt a systematic study of the advantages and the limitations of using these new reagents in bioassays, the different assay formats for individual and multiplexed detection, and the capability of these assays in analyzing real samples.

A Brief History of Mass Spectrometry

This is really an excellent historical brief of mass spectrometry.

http://pubs.acs.org/cgi-bin/sample.cgi/ancham/2008/80/i15/html/ac8013065.html

Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes*,S

Anthony Gonçalves^a,b,c,d, Emmanuelle Charafe-Jauffret^c,e,f, François Bertucci^b,c,f, Stéphane Audebert^a, Yves Toiron^a, Benjamin Esterni^g, Florence Monville^f, Carole Tarpin^b, Jocelyne Jacquemier^e,f, Gilles Houvenaeghel^c,h, Christian Chabannon^c,i, Jean-Marc Extra^b, Patrice Viens^b,c, Jean-Paul Borg^a,c,j and Daniel Birnbaum^f,j

Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to “luminal-like” cell lines and to “basal-like” cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values.

http://www.mcponline.org/cgi/content/short/M700487-MCP200v1

Targeting the oncogene and kinome chaperone CDC37

a new target for cancer therapy.

Nature Reviews Cancer 8, 491-495 (July 2008) | doi:10.1038/nrc2420

 

Abstract

CDC37 is a molecular chaperone that physically stabilizes the catalytic domains found in protein kinases and is therefore a wide-spectrum regulator of protein phosphorylation. It is also an overexpressed oncoprotein that mediates carcinogenesis by stabilizing the compromised structures of mutant and/or overexpressed oncogenic kinases. Recent work shows that such dependency of malignant cells on increased CDC37 expression is a vulnerability that can be targeted in cancer by agents that deplete or inhibit CDC37. CDC37 is thus a candidate for broad-spectrum molecular cancer therapy.