Microarrays go mainstream

Gershon, Diane

doi:10.1038/nmeth1204-263

Cited by 23 publications

(16 citation statements)

References 7 publications

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“…There is an increasing need for rapid and inexpensive detection of a large number of biomolecules. For example, protein arrays can contain tens or hundreds of spots 1–3. Testing of thousands of samples are performed for drug discovery using high throughput screening (HTS) 4,5.…”

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Enhanced Fluorescence of Proteins and Label-Free Bioassays Using Aluminum Nanostructures

2009

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We report the enhanced intrinsic fluorescence from several proteins in proximity to aluminum nanostructured surfaces. Intrinsic fluorescence in proteins is dominated by the tryptophan residues. Intensities and lifetimes of several proteins with different numbers of tryptophan residues assembled on the surfaces of quartz or aluminum nanostructured films were measured. Immobilized protein molecules on the surface of an aluminum nanostructured film resulted in a significant fluorescence intensity enhancement (up to 14-fold) and lifetime decrease (up to 6-fold) compared to the quartz substrates. These large spectroscopic changes allow design of label-free bioassays where detection of binding interactions between proteins can be observed in the presence of a bulk sample solution. Binding of streptavidin to the biotinylated aluminum surface was demonstrated in the presence of 100 µg/mL bovine serum albumin in the sample solution by measurements of tryptophan intensity and lifetime changes.Fluorescence detection is a central technology in analytical chemistry, clinical chemistry, drug discovery, proteomics, genomics, and biochemical research. Almost without exception, fluorescence detection is accomplished using extrinsic fluorophores which are used to label the biomolecules. There is an increasing need for rapid and inexpensive detection of a large number of biomolecules. For example, protein arrays can contain tens or hundreds of spots. 1 -3 Testing of thousands of samples are performed for drug discovery using high throughput screening (HTS).4 , 5 Because of the added complexity of labeling on traditional fluorescencebased bioassays, there is a growing interest in optical methods which provide label-free detection (LFD),6 -8 such as surface plasmon resonance (SPR)9 , 10 or Raman scattering.11 , 12 Also, efforts are currently underway for the direct measurement of the native fluorescence of proteins to eliminate the problems of external tagging in many biological applications. [13][14][15] It is difficult to use intrinsic fluorescence of proteins for specific assays because almost all proteins display tryptophan emission. Additionally, there is typically a high background emission due to the UV absorption and emission wavelengths of 280 and 350 nm, respectively.During the past several years, there have been significant efforts in utilizing the metallic nanostructures or nanoparticles for improved detection of fluorescence.16 -22 This approach represents a fundamental change in fluorescence technology because the fluorophores can be excited by the near fields created by plasmons on the metallic structures, rather than freely propagating light. Additionally, the metallic structure can substantially modify the rates of spontaneous emission and the directionality of the emission. It has been shown that the fluorescence intensity of a number of probes can be increased by proximity to metal island films or nanoparticles. We referred to this phenomenon as metal-enhanced fluorescence © XXXX American Chemical Society ...

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Enhanced Fluorescence of Proteins and Label-Free Bioassays Using Aluminum Nanostructures

2009

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“…Gene expression profiles have been used to classify tumours (Bittner et al, 2000;Ramaswamy et al, 2001;Selaru et al, 2002;Shipp et al, 2002), study the biology of tumour progression and metastasis (Birkenkamp-Demtroder et al, 2002;Ramaswamy et al, 2003), predict clinical outcomes Huang et al, 2003;Iizuka et al, 2003), classify drug resistance (Hofmann et al, 2002;Chang et al, 2003) and identify novel drug targets (Marton et al, 1998). As the technology matures, it is pushing towards mainstream clinical application (Gershon, 2004;Jarvis and Centola, 2005;Cardoso et al, 2008). Surmountable challenges remain, such as standardisation and validation of the competing platforms and analysis techniques.…”

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Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification

et al. 2009

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Tumour stroma gene expression in biopsy specimens may obscure the expression of tumour parenchyma, hampering the predictive power of microarrays. We aimed to assess the utility of fluorescence-activated cell sorting (FACS) for generating cell populations for gene expression analysis and to compare the gene expression of FACS-purified tumour parenchyma to that of whole tumour biopsies. Single cell suspensions were generated from colorectal tumour biopsies and tumour parenchyma was separated using FACS. Fluorescence-activated cell sorting allowed reliable estimation and purification of cell populations, generating parenchymal purity above 90%. RNA from FACS-purified and corresponding whole tumour biopsies was hybridised to Affymetrix oligonucleotide microarrays. Whole tumour and parenchymal samples demonstrated differential gene expression, with 289 genes significantly overexpressed in the whole tumour, many of which were consistent with stromal gene expression (e.g., COL6A3, COL1A2, POSTN, TIMP2 ). Genes characteristic of colorectal carcinoma were overexpressed in the FACS-purified cells (e.g., HOX2D and RHOB ). We found FACS to be a robust method for generating samples for gene expression analysis, allowing simultaneous assessment of parenchymal and stromal compartments. Gross stromal contamination may affect the interpretation of cancer gene expression microarray experiments, with implications for hypotheses generation and the stability of expression signatures used for predicting clinical outcomes.

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“…Chip-technology has become the prevalent method for investigation of gene expression [85,86]. Its current applicability, however, is restricted by the large amounts of sample required.…”

Section: Ultrasensitive Bioanalysismentioning

confidence: 99%

Single Molecule Bioanalysis

Hesse¹,

Schütz

2007

CPA

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Beyond doubt, nanobioscience represents one of the most innovative concepts in current basic and applied research. Technological advances provided the essentials for this ongoing scientific revolution, by making possible the observation, investigation and manipulation of biomaterials down to single molecules. Time-resolved single molecule detection has become the method of choice to characterize structural transitions between different functional states of isolated biomolecules. The development of ultra-sensitive detection schemes also has a strong impact on bioanalysis, as the sensitivity of biochemical assays could be dramatically increased. Whenever the available amount of sample is the limiting factor for unambiguous diagnosis e.g. in medical diagnostics, bioanalytics with single molecule sensitivity can be expected to become even an enabling technology. In this review, we describe methodological requirements for ultra-sensitive detection, and point out major advantages, in particular the novel information content.

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Microarrays go mainstream

Cited by 23 publications

References 7 publications

Enhanced Fluorescence of Proteins and Label-Free Bioassays Using Aluminum Nanostructures

Enhanced Fluorescence of Proteins and Label-Free Bioassays Using Aluminum Nanostructures

Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification

Single Molecule Bioanalysis

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