A Novel Ultraviolet Assay for Testing Side Reactions of Carbodiimides

Wrobel, Nadia; Schinkinger, Manfred; Mirsky, Vladimir M.

doi:10.1006/abio.2002.5646

Cited by 36 publications

(41 citation statements)

References 8 publications

(7 reference statements)

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“…7,27 An example trajectory measured in an active phase is shown in Figure 3c and d, after baseline correction for clarity (see raw trajectories and baseline fit in Supplementary Figure 10). 28 The trajectory shows rapid two-state RTN activity, in which dwell times with submillisecond duration can be resolved (see Figure 3d).…”

Section: Real-time Detection Of Sequential Chemical Reactionsmentioning

confidence: 99%

Single-Molecule Reaction Chemistry in Patterned Nanowells

et al. 2016

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A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K+) versus sodium ions (Na+). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time.

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Section: Real-time Detection Of Sequential Chemical Reactionsmentioning

confidence: 99%

Single-Molecule Reaction Chemistry in Patterned Nanowells

et al. 2016

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“…The subsequent treatment with NHS gives the NHS-ester intermediate, which is more stable in aqueous media, but is less reactive than the O-acylisourea intermediate and also somewhat larger in size. 42,51 The hydrolysis rates will be different for these intermediates present in solution versus immobilized on a surface, and will also depend on pH and, 52 if immobilized, on the structure of the surface monolayer as it impacts their reactivity/ accessibility. 53 The half-life for hydrolysis of EDC in the presence of acetate has been reported to be pH dependent, with the rate 8x faster at pH 4 than at pH 7, and with the hydrolysis half-life being 806 s (3 mM EDC, 50 mM acetate, pH 7).…”

Section: Resultsmentioning

confidence: 99%

“…53 The half-life for hydrolysis of EDC in the presence of acetate has been reported to be pH dependent, with the rate 8x faster at pH 4 than at pH 7, and with the hydrolysis half-life being 806 s (3 mM EDC, 50 mM acetate, pH 7). 52 It is known that the NHS-ester intermediate is prone to side reactions to form byproducts such as an anhydride or an N-acylurea. 42,51 A study of the hydrolysis of NHS-ester terminated SAMs found that the hydrolysis rate can be as much as 1000 times slower for the monolayer than for analogous compounds in solution.…”

Section: Resultsmentioning

confidence: 99%

Characterization of protein immobilization on nanoporous gold using atomic force microscopy and scanning electron microscopy

et al. 2011

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Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize a highly efficient approach for protein immobilization on NPG using N-hydroxysuccinimide (NHS) ester functionalized self-assembled monolayers on NPG with pore sizes in the range of tens of nanometres. Comparison of coupling under static versus flow conditions suggests that BSA (Bovine Serum Albumin) and IgG (Immunoglobulin G) can only be immobilized onto the interior surfaces of free standing NPG monoliths with good coverage under flow conditions. AFM is used to examine protein coverage on both the exterior and interior of protein modified NPG. Access to the interior surface of NPG for AFM imaging is achieved using a special procedure for cleaving NPG. AFM is also used to examine BSA immobilized on rough gold surfaces as a comparative study. In principle, the general approach described should be applicable to many enzymes, proteins and protein complexes since both pore sizes and functional groups present on the NPG surfaces are controllable.

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“…Furthermore, treatment with acidified methanol may lead to solvolysis of acid-labile residues. Unwanted side reactions often occur during carbodiimide-based esterification reactions 26 making it difficult to isolate the desired product.…”

Section: Discussionmentioning

confidence: 99%

“…26 Pectins are only sparingly soluble in the organic solvents used in the first two reactions, and complete methyl-esterification is often difficult to achieve. Furthermore, treatment with acidified methanol may lead to solvolysis of acid-labile residues.…”

Section: Discussionmentioning

confidence: 99%