Homogeneous Protein Analysis by Magnetic Core–Shell Nanorod Probes

Abstract

Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable labelfree homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 -sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

“…1a). The 2 nd Abs attach to the nanoprobe-bound analyte molecules, and, when extending the protein corona thickness, retrieve the concentration dependent measurement signal that has previously been observed for spiking analyte molecules alone into buffer solutions 10 . The exact composition and structure of the protein corona formed on top of the nanoprobes cannot be answered here, while in principle we assume a single layer of adsorbed proteins.…”

“…Afterwards, the NRs were transferred to aqueous solutions by over-coating them with an amphiphilic polymer as described earlier 10, 21 . The functionalization of the NRs with antibodies (Abs) was achieved by carboxy-amine linker chemistry.…”

“…To that end, we employed EDC (N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride) and S-NHS (N-hydroxysulfosuccinimide sodium salt) at ratios of 1 × 10 4 EDC molecules and 3 × 10 4 S-NHS molecules per NR. The NR concentration was determined by inductively coupled plasma mass spectrometry and by the geometric NR dimensions determined by transmission electron microscopy as described elsewhere 10 . We employed a NR concentration of 730 pM and 2-(N-morpholino)-ethanesulfonic acid (MES) buffer solution at a concentration of 50 mM and at a pH of 5.5 throughout the whole functionalization procedure.…”

“…Measurements were conducted at a RMF amplitude of 10 mT and a rotational frequency of 1 kHz. These parameters are chosen for optimal measurement signals to detect target proteins by our specific nanoprobes 10, 24 . Phase lag differences were recorded with respect to a suitable reference value.…”

“…When applying linearly polarized incident light, this effect allows for deducing the actual nanoprobe orientation in the sample solution via transmission measurements. By correlating the measured actual nanoprobe orientation with the momentary vector of the applied RMF, the phase lag angle can be determined, and our signal is defined by the change in phase lag angle (Δα) between the sample and a suitable reference 10–12. Next to the inherent advantages of homogenous magnetic nanoparticle-based measurement methods, we show that our method is capable of determining quantitative biomarker concentration levels in complex samples by in - situ referencing.…”