Amine-modified surface

The phosphate backbone of DNA molecules often results in undesirable electrostatic interactions with the substrate. Although the electrostatic interactions of DNA can be utilized for physical adsorption of DNA to the surface, this process can also lead to the nonspecific physical adsorption of target DNA on the surface. Rather than sample DNA hybridizing to the probe, it can adsorb to the surface and lead to interferences with the final detection call. Nonspecific adsorption effects have primarily been examined by the microarray community. Blocking strategies have been developed to prevent these nonspecific interactions. Succinic anhydride (SA) and bovine serum albumin (BSA) are two common methods to prevent nonspecific adsorption on amine modified surfaces. Blocking strategies are desired to react with or pas-... 

Figure 2 Schemes of the most commoniy used immobiiization techniques. (1) Coupiing of aminopropyi functionaiity to a siiica-based support, (2) Giutaraidehyde coupiing, activation of amine-modified surface with giutaraidehyde, (3) immobiiization of enzyme on giutaraidehyde-activated support, (4) Carbodiimide coupiing, (5) Aryiamine support prepared by p-nitrobenzoyi chioride with alkyla-mine-modified support foiiowed by reduction (6) and (7) Enzyme immobiiization by azo coupiing through the tyrosine moiety to the aryiamine after diazotization.

First, a reactive comonomer unit can be introduced for a direct covalent immobilisation. When the material surface to be coated contains hydroxyl groups (e.g., glass, silicon, titanium alloys), the silyl-hydroxyl reaction can be used. Following this route, Yang et al. (2010) combine A-isopropyl acrylamide (NiPAAm) with a 3(trimethoxysi-lyOpropyl methacrylate unit to provide a stable thermo-responsive cell culture surface on glass carriers. On an amine-modified surface, a randomly distributed copolymer of... 

Key words Surface force - amine-modified surface - long-range attraction - atomic force microscope - isoelectric point... 

Fig. 1 Profiles of interaction forces between amine-modified surfaces at various pHs in 1 mM NaBr aqueous solution at the room temperature. pH was adjusted by adding HNO3 or NaOH. The isoelectric point varies depending on the preparation, and has been obtained to be 6 for this sample. The van der Waals attraction calculated using nonretarded Hamaker constants of 1.0x10 ° (solid line) and 0.4 X 10 ° J (dashed line), respectively are also plotted...

Fig. 2 Salt concentration dependence of the decay length (Do) of the long-range attraction between amine-modified surfaces. Data were taken for samples exhibiting attraction at different pHs (pis ) pH 9 (1), pH 5.6 (m and n), pH 6 (o). Debye length is also included as a reference (dotted line)...

Smith and co-workers (194) have used this chemistry to prepare carboxyl-modified Si(lll) surfaces at which polylysine-tethered DNA is electrostatically adsorbed (Fig. 60). An alternative approach involved covalent attachment of a pre-synthesized oligonucleotide bearing a terminal carboxyl group to an amine-modified Si(001) surface (195). 

A schematic representation of the formation of PAA/PAH NPSs is shown in Figure 7.7. The BMS particles are modified with a layer of 3-aminopropyltriethox-ysilane (APTS) on the BMS skeleton. This process introduces amine (—NH2) surface... 

SIAB and sulfo-SIAB have been used to make a high-capacity RNA affinity column for the purification of human IRP1 and IRP2 (Allerson et al., 2003), to couple antibodies or Fab fragments to amine-modified microparticles (Harma et al., 2000), and in the attachment of oligonucleotides to surfaces for detection arrays (Adessi et al., 2000). 

Hong et al. (2004) also found that modification of PAMAM dendrimers with a short PEG linker arm could act to reduce nonspecificity caused by the amines on the dendrimer-modified surface. An azido-PEGj-aininc spacer was activated with nitrophenyl carbamate to yield an activated intermediate that could be used to modify the amines on the dendrimer (Figure 7.24). Reaction at high molar ratio resulted in about 61 PEG-azido spacers on the dendrimer. Reduction of the azido group to an amine using triphenylphosphine in THF provided the dendrimer-PEG-amine derivative for surface modification. The added presence of the PEG spacer arm reduced... 

Figure 13.4 APTS-modified surfaces may be further derivatized with amine-reactive crosslinkers to create additional surface characteristics and reactivity. Modification with NHS-PEG4-azide forms a hydrophilic PEG spacer terminating in an azido group that can be used in a click chemistry or Staudinger ligation reaction to couple other molecules.

Figure 13.6 APTS-modified surfaces can be activated with DSC to form amine-reactive succinimidyl carbonates for coupling proteins or other amine-containing molecules.

An amine-containing surface, such as an APTS-modified glass slide (see Chapter 13, Section 2), may be modified with a long-chain NHS-salicylic acid methyl ester derivative (Lonza), which then can be converted to the SHA group for coupling to a P(D)BA-modified protein. The following protocol describes this method. 

Figure 18.5 The modification of an APTS-modified surface containing amines with bis-NHS-PEG5 yields hydrophilic spacers containing terminal NHS esters for coupling proteins.

What is the reason for the overwhelming acceptance of stationary phases based on high-purity silicas in the pharmaceutical industry The answer is simple superior peak shapes for analytes with basic functional groups, which has been a problem with older phases. The older, low-purity silicas contain metal ions buried in the matrix of the silica. These contaminants acidify the surface silanols, and the consequence is a strong and non-uniform interaction with basic analytes. This in turn results in tailing peaks, which is an impediment for accurate peak integration and peak resolution. Of course, adding appropriate additives, such as amine modifiers, to the mobile phase can solve these difficulties. But this is an unnecessary and undesired complication in methods development. Therefore, silicas that are free from this complication are much preferred. 

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