Probe density

Stirling and Graff (1995) preferred treatment with sodium metaperiodate followed by heating on citrate buffer. Sections were treated by heating them with a hotplate at 95°C while they floated on 0.01 M citrate buffer (pH 6.0). Using this combination, the authors reported a high probe density and sections remained intact, with good ultrastructural detail. 

Dendrimers are branched chemical structures which can possess a range of terminal functionalities. Covalent attachment of dendrimers to a support builds a 3D structure along its surface which can subsequently be grafted with ligands which, as a consequence of the larger surface area, can yield higher probe densities. 

Membranes have been developed that possess improved characteristics with respect to their lateral wicking and spot resolution. The best example of this type of material is anodically oxidized alumina whose structure consists of pores, with very little material forming the walls, possessing a surface area ratio of approximately 500 f. The benefits of this material are its higher sensitivity (more immobilized capture probe) and higher probe densities. 

The number of probe molecules per square millimeter defines the probe density of a spot. Probes within an element or spot may have densities on the order of 10 to -10 molecules/mm depending upon the molecular size of the nucleic acid (e.g., short oligonucleotide vs. cDNA). 

The highest efficiency for probe attachment was found for the reaction of carboxylated probe with an aminosilane surface. However, the level obtained by simple adsorption (without the addition of ED AC) was equally as high (mean probe density = 600 to 700 fmoles/cm ). Conversely, the attachment of an amine-modified probe to a carboxyl surface was much less efficient (mean probe density = 300 fmoles/cm ). The least efficient coupling was obtained using aldehyde surfaces (mean probe density = 150 fmole/cm ). 

In an earlier study from Ken Beattie s group (then at the Houston Advanced Research Center), a similar conclusion was reached using a 9-mer oligonucleotide and triethylene glycol phosphoryl repeat unit as the spacer (1995). An optimal probe density leading to the greatest hybridization... 

Figure 3.14 Probe density vs. hybridization efficiency. (From Beattie, W.G. et al.. Mol. Biotechnol, 4, 213-225, 1995. Copyright, 1995 Humana Press, Inc. With permission.)...

Fig. 16 Response of a biosensor versus analyte concentration as described by (1). The signal density (in arbitrary units) depends on the Kq value of the capture probes [dark curves (high capture probe density) vs. light curves (low capture probe density)], and on the capture probe density [red curves (low Kq) vs. blue curves (high ATd)]. The green horizontal line represents the threshold signal density required to obtain a signal distinguishable from noise. High capture probe density and high Kq (dark blue curve) can result in lower limits of detection than low capture probe density and low Kv (light red curve)...

A multi-layer surface architecture composed of SAM/streptavidin/probe was employed for the hybridization study (Fig. 19). Since the streptavidin density on the functional stripes was identical to that on a homogenous surface [9], i.e., 2.2 x 1012 molecules cm-2, the probe density was estimated to be 2.9 x 1012 molecules cm 2 by knowing from the diffraction signal the binding stoichiometry between streptavidin and the probe (ca. 0.75) [16],... 

Peterson AW, Heaton RJ, Georgiadis RM (2001) The effect of surface probe density on DNA hybridization. Nucleic Acids Res 29 5163-5168... 

The density of tethered redox probe at the surface is another useful parameter in electroactive assembly characterization. The amount of redox probe corresponds to the Faradaic charge of the redox process and is measured from the voltammogram using the area ij E under the voltammetric wave. The area is readily converted to the redox probe density (F, in mol cm -) [47]. For well-ordered -alkanethiol SAMs, the total (electroactive plus diluent) surface density is one molecule of adsorbate per 20 A, or about 8 x 10 mol cm [38]. To minimize disorder and electrochemical communication between tethered redox probes, the amount of redox probe should not exceed 10 20 % of the total adsorbate [23]. 

Most of these methods are applied to either PCR products or presynthesized oligonucleotides in combination with liquid-spotting systems. This allows control and optimization of the amount of the immobilized singlestrand [149]. SH-coupling or benzaldehyde-semicarbazide-coupling delivered maximal densities of approximately 10-100 pmol/cm [160,161], whereas binding of amino-functionalized PCR products to an aldehyde glass surface resulted in only 600 fmol/cm [154], However, only 10-30% of the immobilized probes took part in the hybridization reaction so that the optimal probe density was even further reduced [149]. The density of spots in these arrays is limited by mechanical constraints of liquid delivery or surface manipulation. 

These potential complications of the quadrupole interaction and broadening due to atomic disorder discouraged the widespread use of solid state NMR as a structural probe for metals. Much of the continuing NMR work directed towards probing densities of states in metals was carried out at low temperatures, a further disincentive to the widespread application of NMR to these materials. Examples of the early use of NMR... 

Figure 3. Melting curves for different surface probe densities (coverage) as indicated. In solution Tm = AH0/(AS0 - RlnC), W = ARTfJAHo. ...

Figure 3. Optical gain as a function of the pump energy density from 50 to 115 mJ/cm with a power probe density of 195 pW/cm ...

Over the last few years, several different types of microarrays of various probe densities and content—DNA or protein—have become commercially available (Table 5.6-1). These provide microarray tools for analyzing samples derived not only from human tissues, but also from a wide spectrum of other species ranging from bacterial pathogens to Arabidopsis to mouse. 

---