AFM-based protein fishing in the pulsed electric field

Abstract

A combination of (atomic force microscopy) based fishing (AFM fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat sur face of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM fishing has been demonstrated using detection of cytochrome b 5 protein. Selec tion of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.

Highly sensitive protein detection by biospecific AFM‐based fishing with pulsed electrical stimulation

“…Another factor influencing the protein fishing efficiency (as is indicated by the results obtained in this study and in ) is an external electric field. To provide the detection efficiency, pulsed electrical stimulation of AFM‐based fishing is required.…”

“…Earlier in our studies [5,12] we have shown that the concentration of proteins on the surface may be attained without turbulent stirring or high-strength fields. We have shown [5] that the concentration is more efficient upon the fast (injection) input of protein solution into the measuring cell.…”

“…The results of our further studies [12] have indicated that the flow system can be used for highly sensitive detection of proteins by the AFM-MS method. In this case, to increase the protein fishing efficiency a pulsed AC electric field can be employed.…”

“…Employing the flow system, cytochrome b 5 protein was detected in 10 À15 M solution upon electrical stimulation; the presence of the protein was not registered without the stimulation. Thus, we have shown that weak electric fields can be used for concentration of the protein [12]. The use of the electric field does not require employment of microcapillarieswhich eliminates the problem of clogging of the microcapillaries in the analysis of biological fluids.…”

“…Pulses of AC voltage had 1 ns rise time, 1 kHz frequency and 500 mV amplitude (nanosecond pulsed AC electric field). It was shown that the efficiency of protein fishing increases upon stimulation with exactly nanosecond pulsed AC electric field . In contrast to the pulsed voltage, the constant electric field (without the applied AC electric field) and AC sine wave electric field did not increase the efficiency of the AFM‐based fishing of cytochrome b 5 .…”