Quantification of DNA and protein adsorption by optical phase shift.
Quantification of DNA and protein adsorption by optical phase shift.
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DOI：
10.1016/j.bios.2009.06.033
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发表时间：
2009-09-15
影响因子：
12.6
通讯作者：
Unlü MS
中科院分区：
文献类型：
--
作者：
Ozkumur E;Yalçin A;Cretich M;Lopez CA;Bergstein DA;Goldberg BB;Chiari M;Unlü MS
与荧光测量相比，无标记检测方法的主要优点是其定量检测能力，因为吸附材料的绝对测量有利于生物分子相互作用的动力学表征。干涉测量技术将光学相位与表面上的生物分子层密度联系起来，但转换因子之前尚未准确确定。我们提出了一种用于相移测量的校准方法，并将其应用于表面结合的牛血清白蛋白、免疫球蛋白 G 和单链 DNA。将已知浓度的生物分子溶解在无盐水中，以精确的体积点在阵列表面上，并在水蒸发后留下易于计算的质量。使用我们的无标记技术，将计算出的生物层质量与测量的厚度进行比较，我们观察到超过 4 个数量级的线性依赖性。我们确定，广泛接受的 1 nm 厚度转换对应于 ~1 ng/mm2 表面密度，对于这些物质来说保持得相当好，并且通过我们的实验现在可以针对不同类型的生物分子进一步指定。通过精确校准厚度对表面密度的依赖性，我们建立了一种关系，可以精确确定单链 DNA 和两种不同蛋白质的分子绝对数量。
A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA. Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to ~1 ng/mm2 surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.
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DOI：
10.1073/pnas.0511214103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Moiseev, L;Ünlü, MS;Cantor, CR
通讯作者：
Cantor, CR
影响因子：
5.4
作者：
Ramachandran, N;Larson, DN;LaBaer, J
通讯作者：
LaBaer, J
影响因子：
3.9
作者：
Elhadj, S;Singh, G;Saraf, RF
通讯作者：
Saraf, RF
影响因子：
7.4
作者：
Pirri, G;Damin, F;Depero, LE
通讯作者：
Depero, LE
DOI：
10.1073/pnas.0711421105
发表时间：
2008-06-10
影响因子：
11.1
作者：
Oezkumur, Emre;Needham, James W.;Uenlue, M. Selim
通讯作者：
Uenlue, M. Selim