Ultrananocrystalline Diamond Membranes for Detection of High-Mass Proteins

Mechanical resonators realized on the nanoscale by now offer applications in mass sensing of biomolecules with extraordinary sensitivity. The general idea is that perfect mechanical mass sensors should be of extremely small size to achieve zepto- or yoctogram sensitivity in weighing single molecules similar to a classical scale. However, the small effective size and long response time for weighing biomolecules with a cantilever restricts their usefulness as a high-throughput method. Commercial mass spectrometry (MS), on the other hand, such as electrospray ionization and matrix-assisted laser desorption and ionization (MALDI) time of flight (TOF) and their charge-amplifying detectors are the gold standards to which nanomechanical resonators have to live up to. These two methods rely on the ionization and acceleration of biomolecules and the following ion detection after a mass selection step, such as TOF. The principle we describe here for ion detection is based on the conversion of kinetic energy of the biomolecules into thermal excitation of chemical vapor deposition diamond nanomembranes via phonons followed by phonon-mediated detection via field emission of thermally emitted electrons. We fabricate ultrathin diamond membranes with large lateral dimensions for MALDI TOF MS of high-mass proteins. These diamond membranes are realized by straightforward etching methods based on semiconductor processing. With a minimal thickness of 100 nm and cross sections of up to $400\times 400\mu {\mathrm{m}}^2$, the membranes offer extreme aspect ratios. Ion detection is demonstrated in MALDI TOF analysis over a broad range from insulin to albumin. The resulting data in detection show much enhanced resolution as compared to existing detectors, which can offer better sensitivity and overall performance in resolving protein masses.

Figure 1

The principle of operation of the diamond nanomembrane detector: (a) schematic of a MALDI TOF mass spectrometer. Proteins are desorbed and ionized by MALDI, followed by a time-of-flight separation of proteins of different mass. (b) Sketch of the detector configuration and operation principle. The detector consists of a freestanding diamond nanomembrane (receiving the kinetic energy of the impacting proteins), an extraction gate, MCP, and an anode. The electric field between the diamond nanomembrane and the extraction gate allows enhanced electron field emission by reducing the energy barrier that electrons from inside the membrane have to overcome. The MCP amplifies the number of electrons that pass through the extraction gate and arrive at the MCP. This flux of electrons is collected in the anode, and the signal is traced on the oscilloscope in the time domain. The inset shows an optical microscope image of the $400\times 400\mu {\mathrm{m}}^2$ freestanding 100-nm-thick $p$-type diamond nanomembrane used for this study. The buckling patterns in the membrane are generated when the compressive residual stress is released during processing.

Figure 2

Measurements with the diamond nanomembrane detector: (a) the schematic setup of the experimental configuration for field emission at room temperature. (b) Obtained data and analytical fits for field emission: the I-V curve of the measurement is given by the red filled circles, while the theoretical fit is shown as a black line. Inset: Log-scale current analysis of the three distinct mechanisms of thermionic emission (THc), Schottky emission (Sc), and Fowler-Nordheim tunneling (FNc) and comparing this to the measurement (Mc) in the intermediate voltage range. (c) and (d) show MALDI TOF mass spectra of insulin (5735 Da) and cytochrome c (12 365 Da), respectively. Insets in (c) and (d): The magnified view of the recorded peaks of insulin and cytochrome c, respectively. The shape of the peaks reveal a sharp onset due to quasidiffusive transport of phonons in the out-of-plane direction (normal to the surface of the membrane) and an exponential decay by the lateral heat diffusion along in-plane direction (across the membrane) as shown in a red dashed line. The rise and decay time constants for insulin are 76.44 and 275.06 ns, respectively, while the peak of cytochrome c shows 73.36 and 265.09 ns. The peaks are very similar for both proteins since the protein concentrations, initial accelerating voltages, and, of course, the detecting nanomembranes are the same. The mass resolution ($m/\mathrm{\Delta }m$) increases rather with increasing molecular mass. The mass resolution ($m/\mathrm{\Delta }m$) obtained for insulin and cytochrome c is approximately 350 and approximately 480, respectively.

Figure 3

Heat flux and temperature profile of the diamond nanomembrane showing a plot of the heat input ($Q_{\mathrm{in}}$) caused by the impact of the proteins and the resulting field emission current $J$ as a function of time (a). Field emission leads to an effective cooling represented by the emitted heat ($Q_{\mathrm{out}}$) in (b) which limits the peak temperature of the membrane to about 800 K. The heating is initially strongest in the center, as shown by the peak in (c) obtained at $t=0.53\mu \mathrm{s}$, which is the onset of the field emission current. After the field emission begins to cool the nanomembrane, the temperature profile flattens in the middle, as depicted in (d) at $t=2\mu \mathrm{s}$, at the tail of the field emission peak, when the peak temperature drops below 800 K.