Quantification of DNA and protein adsorption by optical phase shift

Abstract

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/mm 2 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.

Integrated imaging instrument for self-calibrated fluorescence protein microarrays

“…This method of monitoring protein binding can quantify the bound density with pg/mm 2 accuracy and precision. 19 Detection using a second antibody offers higher sensitivity and specificity than label-free detection of analytes. Labelfree methods lack an additional step to isolate specific binding from non-specific binding.…”

Spectral Self-Interference Fluorescence Microscopy to Study Conformation of Biomolecules with Nanometer Accuracy

“…Deposition of DNA and protein molecules on the surface could change the refractive index, and we should regard the change of thickness with caution. Often lower surface density of molecules results in lower refractive index, and for our cases we can assume the refractive index of the biomaterial to be close to that of SiO 2 [70][71][72][73][74]. However, the additional amount of biomaterial on the surface can always be quantified accurately by the change of optical path length.…”

Surface chemistry and morphology in single particle optical imaging

“…Label-free optical imaging biosensors are those that use imaging optics to rapidly measure a large sensor surface area. In this section, we describe three members of the family of label-free optical imaging biosensors, which are darkfield microscopy (DF/TIRM), surface plasmon resonance imaging (SPRi), and interference reflectance imaging (IRIS, iSCAT) [31][32][33][34]. We also introduce the notion that for label-free imaging approaches, even more than for other types of label-free sensors, the surface morphology of the sensor's functionalization is extremely important.…”

“…Interference reflectance imaging has been utilized in several label-free biosensor designs, namely, by the Gauglitz group as Reflectance Interference Spectroscopy (RIfS), Sandoghdar group as Interferometric Scattering Microscopy (iSCAT), and Ünlü group as the Interference Reflectance Imaging Sensor (IRIS) [34,52,53]. All of these techniques measure the binding of biomolecules as small changes in the reflectivity or reflectivity spectrum of a layered substrate.…”

“…Wide-field coherent light imaging is nontrivial because of long-range interference effects and laser speckle -to overcome these, a ground-glass diffuser with temporal averaging may be used to ameliorate these effects, provided the exposure time is sufficiently long [34]. A second option is to limit detection to dynamic measurements, in which sequential images are subtracted pixelwise, yielding highly sensitive detection of small changes in what is an otherwise highly nonuniform illumination field [53].…”