Recording intracellular (IC) bioelectrical signals is central to understanding the fundamental behaviour of cells and cell networks in, for example, neural and cardiac systems1,2,3,4. The standard tool for IC recording, the patch-clamp micropipette5 is applied widely, yet remains limited in terms of reducing the tip size, the ability to reuse the pipette5 and ion exchange with the cytoplasm6. Recent efforts have been directed towards developing new chip-based tools1,2,3,4,7,8,9,10,11,12,13, including micro-to-nanoscale metal pillars7,8,9, transistor-based kinked nanowires10,11 and nanotube devices12,13. These nanoscale tools are interesting with respect to chip-based multiplexing, but, so far, preclude targeted recording from specific cell regions and/or subcellular structures. Here we overcome this limitation in a general manner by fabricating free-standing probes in which a kinked silicon nanowire with an encoded field-effect transistor detector serves as the tip end. These probes can be manipulated in three dimensions within a standard microscope to target specific cells or cell regions, and record stable full-amplitude IC action potentials from different targeted cells without the need to clean or change the tip. Simultaneous measurements from the same cell made with free-standing nanowire and patch-clamp probes show that the same action potential amplitude and temporal properties are recorded without corrections to the raw nanowire signal. In addition, we demonstrate real-time monitoring of changes in the action potential as different ion-channel blockers are applied to cells, and multiplexed recording from cells by independent manipulation of two free-standing nanowire probes.
Separation of a nanoelectronic detector element from much larger interconnections is necessary for internalization of the detector without damaging the cell of interest1,2,3,10. To date, all approaches7,8,9,10,11,12,13,14 have focused on fabricating nanodevices on planar substrates, where the detector protrudes from the surface and target cells are brought into contact with the nanodevices by direct seeding and culture7,8,9,14 or manipulation of a culture substrate10,11,12,13 (Fig. 1a). These studies enabled the demonstration of new nanodevice concepts and multiplexed detection, but also have limitations, including (1) device positions are determined during chip fabrication and cannot be reconfigured during an experiment, (2) it is difficult to target specific cells or subcellular regions and (3) minimally invasive in vivo measurements are difficult. In comparison, a free-standing probe that can be manipulated in three dimensions (3D; Fig. 1b) would allow targeting of specific cells cultured on substrates or within tissue, although the size of manipulator for such probes will limit multiplexing compared to chip-based methods2,3. In this regard, development of a general strategy to present nanoelectronic device elements, such as the kinked silicon nanowire field-effect transistor (nanoFET)10, in a free-standing probe structure could expand substantially the capabilities and applications of these devices in electrophysiology.
The realization of free-standing probes with nanoelectronic device tips requires bridging length scales from the nanoscopic to the macroscopic in a manner that yields robust electrical and mechanical properties. We focus on meeting these challenges for kinked silicon nanowire nanoFET tips as a general example of a two-terminal active nanoelectronic device. In this case, the nanowire arms of the kinked structure serve as nanoscale connections that must be electrically and mechanically connected to the macroscopic handle that serves as input/output to measurement electronics. Our free-standing kinked nanowire probe fabrication involves two overall stages (Fig. 2): (1) lithographic patterning of a nanometre-to-millimetre probe end and (2) mechanical assembly of the probe end to a millimetre-to-macroscale probe body.
Key steps in the probe-end fabrication are as follows (Fig. 2a–c, Supplementary Methods). First, kinked Si nanowires with nanoFETs encoded synthetically at the kink tip15 were deposited selectively on a substrate coated with sacrificial nickel and SU8 photoresist layers (Fig. 2a). A representative optical image (Fig. 2d) shows the resulting kinked nanowire and lithography alignment markers. Second, electron-beam lithography (EBL) and photolithography (PL) were used to define the kinked nanowire tip region and the probe body in the SU8 layer, respectively (Fig. 2b). Figure 2e shows the kinked nanowire region after these steps. Metal interconnects and top SU8 passivations were fabricated, and a photosensitive protection cap was defined at the tip (Fig. 2c). This cap protects the nanowire tip from capillary forces and contamination during assembly to the probe body and storage prior to cell experiments. Images of a completed probe-end structure (Fig. 2f) show the overall probe structure and protection cap at the end of the wired-up nanowire tip. The probe-end structures are easily fabricated in parallel. We processed 3 × 4 arrays of probes on substrates of about 3 × 3 cm2 (Supplementary Fig. 1), and larger arrays could be fabricated using EBL instruments capable of handling larger substrates.
To complete the probe we assembled the probe-end structure to a printed circuit board (Fig. 2g, and Supplementary Methods). A silicon microlever shorter than the nanowire/SU8 probe structure was glued to the printed circuit board, and then the probe-end structure, released by Ni underlayer etching, was removed from solution (Fig. 2h), aligned, glued and electrically connected to the microlever/printed circuit board. An image of an assembled probe (Fig. 2i) highlights the electrical connection and microlever support; a zoom of the tip region (Fig. 2j) shows the nanowire/SU8/metal interconnect structure and protection cap of the probe end.
We used the free-standing nanowire probes alone or with a second independent probe to interrogate live cells (Fig. 3a). All measurements were carried out in an inverted microscope with probes mounted in XYZ manipulators and a temperature-regulated cell medium (Fig. 3b). Prior to measurements, the photosensitive protection cap on the end of the nanowire probe was removed in solution, and for cell measurements the nanowire end was coated with phospholipid layers, as described previously10,11,12 (see Supplementary Methods).
A differential interference contrast image that shows the ends of a free-standing nanowire probe and patch-clamp micropipette recording from a single cultured cardiomyocyte cell (Fig. 3c) highlights the capability of targeting the nanowire probe to specific cell regions and its smaller tip size compared to a patch-clamp micropipette. The sensitivity of these nanoFET probes was characterized before and after cell measurements so that recorded conductance data could be presented as potential (millivolts) values (see Supplementary Methods). Representative data recorded in PBS solution (Fig. 3d) yield a sensitivity (7,730 nS V−1) similar to previous values for chip-based kinked nanowire devices10 and to the average (8,500 ± 4,300 nS V−1) for probes in our studies. Significantly, the variation in nanoFET sensitivities before and after cell measurements was <10% and often only ∼1%, which shows that the probes provide reproducible, quantitative potential data.
Data recorded with a phospholipid-modified free-standing nanowire probe from spontaneously beating rat neonatal cardiomyocytes (Fig. 3e) exhibit regular peaks with amplitude (67 mV), duration (260 ms) and shape characteristics16,17 of IC cardiac action potentials (APs). The IC AP peaks were observed 1–20 s after the phospholipid-modified nanoFET was brought into gentle contact with the cell membrane using the 40 nm step resolution of the manipulator, and they disappeared when the probe was retracted from the membrane. We were unable to record IC AP signals with unmodified nanoFET probes. These results are consistent with biomimetic membrane fusion10,11,12,13,18, and the high positioning accuracy of our free-standing probes should enable future studies of targeting and internalization with specific ligand/receptor functionalized19,20,21 probe tips.
In addition, comparison of IC AP peaks recorded sequentially from three cells using the same nanowire probe (Fig. 3f) shows that these similar AP signals have amplitudes of 51, 46 and 56 mV for cells 1, 2 and 3, respectively. These recorded AP amplitudes are consistent with the average values determined from independent nanoFET and patch-clamp probes, 55 ± 16 mV (N = 15) and 58 ± 25 mV (N = 13), respectively, on similar cultured cardiomyocytes (DIV3). These results show that our free-standing nanowire probes can be used in multiple measurements on arrays of cells or cell networks, which could improve the efficiency of such studies compared to patch-clamp measurements where the glass pipette is replaced for each try on a new cell.
We also carried out simultaneous measurements on the same cell using both kinked nanowire and patch-clamp probes (for example, Fig. 3a,c). In these experiments, we first established IC recording with a patch clamp in the whole-cell current-clamp mode, and then brought the kinked nanowire probe into contact with the cell to establish the nanoFET IC signal. These data exhibit several key features. First, qualitative inspection of the simultaneous IC AP signals from the nanoFET and patch clamp (Fig. 3g) show that they are very similar in absolute amplitude and time-dependent shape. Second, analysis of the signal changes as the nanoFET enters the cell (Fig. 3h) reveals that there was a about a −50 mV baseline jump as the nanoFET crosses the cell membrane (triangle, Fig. 3h), and that, after this first entry peak, all subsequent AP signals from the nanoFET and patch clamp (blue and red, respectively, Fig. 3h) overlap identically with a 65 mV peak amplitude.
A small depolarization (∼8 mV) in the resting potential of the patch clamp was observed as the nanowire probe entered the cell, which is probably because of leakage of the patch electrode membrane seal and not nanoFET leakage, as follows. First, the patch-clamp recording is sensitive to the contact between the nanoFET and cell membrane, and we have observed that touching but not penetrating cell membranes with the nanoFET can lead to depolarization and/or loss of patch-clamp signals. Second, independent single-probe experiments show that the mean stable AP recording time for the nanoFET probe, five minutes (N = 10), is longer than that for the patch-clamp probe, 2.4 minutes (N = 11), using the same experimental set-up. Third, extended recording with a nanoFET probe (Supplementary Fig. 2) yields full-amplitude 85 mV AP signals over a five minute period with <3% loss of signal amplitude, and thus demonstrates a high-quality membrane seal and minimal effect on the physiological status of the cell by nanoFET insertion.
Significantly, only two parameters intrinsic to the nanoFET are used to convert probe conductance into local potential: (1) the extracellular conductance baseline, which is assigned zero potential (black arrow, Fig. 3h), and (2) the sensitivity of the nanoFET. Hence, the agreement of both position and shape between the two traces is the first and only direct evidence to date for minimally invasive IC recording by a nanodevice. Moreover, these data clearly differentiate our nanoelectronic probes from other IC-like recordings based on high seal resistance around extracellular electrodes and localized electroporation7,8,9,22. In addition, the average amplitude of the APs, 55 ± 16 mV (nanoFET, N = 15) and 58 ± 25 mV (patch clamp, N = 13), and rest potentials, −37 ± 10 mV (nanoFET) and −43 ± 13 mV (patch clamp), obtained from single-probe recording experiments are indistinguishable statistically. Last, the highest amplitudes of APs reach 90 mV (Supplementary Fig. 3), which thus confirms the high quality of the nanoFET/cell membrane junctions.
Our free-standing kinked nanowire probes were also used to characterize quantitatively the effects of ion-channel blockers on recorded IC APs. First, the L-type Ca2+-channel blocker nifedipine was injected into the medium after a stable IC AP recording was established from a cardiomyocyte cell. Monitoring of the nanoFET signal (Fig. 3i) shows a constant AP peak amplitude and progressive decrease in the full-width at half-maximum (FWHM) of 147, 130 and 102 ms at times 0, 60 and 110 s, respectively, after nifedipine injection. The decrease in AP peak FWHM at a constant peak amplitude is consistent with a decrease in the Ca2+ current caused by nifedipine binding23. Second, addition of the Na+ channel blocker tetrodotoxin (TTX) monitored in a separate experiment (Fig. 3j) shows a rapid decrease in the initial fast rising (depolarization) edge and corresponding decrease in the peak amplitude of the AP versus time. The slopes (V s−1)/peak amplitudes (mV) were 3.42/44, 1.73/28, 0.78/20 and 0.53/13 for 0, 3, 12 and 22 s, respectively, after TTX addition, and are consistent with the suppression of the inward Na+ current caused by TTX binding23. These results confirm that nanoFET probes record details of IC AP changes in a reliable and robust manner, and show that they can be a tool for drug screening and cell signalling studies in the future.
Last, we explored the use of two nanoFET probes in multiplexed recording experiments. For example, two distinct nanoFET probes mounted on independent manipulators (Fig. 4a) were used to target cultured cardiomyocyte cells precisely, including two adjacent cells with well-defined alignment (Fig. 4b) and a single cell (Fig. 4c). Targeting with submicron resolution was readily achieved using differential interference contrast imaging, and could be improved further using fluorescence imaging. Representative data recorded from two nanoFET probes as they were sequentially brought into contact and internalized by two adjacent cardiomyocytes (Fig. 4d) highlight several key points. First, following gentle contact of the phospholipid-modified probes with the cell (40 nm step resolution), both probes showed a short (∼2 s) time delay before IC AP peaks appeared, and stable full-amplitude APs developed after several additional seconds. Second, the full-amplitude APs recorded with probe-1 and probe-2 (50 and 45 mV, respectively) are consistent with both independent patch-clamp measurements and the literature24 for neonatal (versus adult) cardiomyocytes. The extracellular-to-IC baseline shifts can also be smaller for these neonatal cells (that is, −20 mV for probe-2 versus −43 mV for probe-1), but are consistent with the stage of our neonatal cell culture24. These multiplexed studies further highlight the robustness of our free-standing nanowire probe fabrication and the potential to characterize AP timing differences for precisely defined nanoprobe/cell configurations and structures too small for conventional patch-clamp measurements, such as dendritic spines25. Moreover, the capability to specify with high resolution the specific cells and/or cellular regions targeted by the nanoFET detectors represents an advantage compared to multiplexed recording with nanoelectronic probes fabricated on planar substrates. The physical dimensions of the manipulators used for targeting nanoFET probes will limit the level of multiplexing compared to chip-based methods2,3, although future studies that incorporate kinked nanowire structures with synthetically encoded multiple nanoFET sensors26 could mitigate this by increasing the number of detectors on each probe.
In summary, we have demonstrated a robust approach for the fabrication of free-standing silicon kinked nanowire probes with encoded nanoFET detectors at the tip ends. These probes were manipulated in 3D with submicron precision to target specific cells and/or cell regions and record stable full-amplitude APs from spontaneously beating cardiomyocytes. Simultaneous measurements from the same cell made with kinked nanowire and patch-clamp probes showed that the same AP amplitude and temporal properties were recorded without corrections to the raw nanowire signal, which thus demonstrates the first direct evidence for a minimally invasive, true IC recording by a nanodevice. In addition, we demonstrated real-time monitoring of AP changes as different ion-channel blockers are added to cells, and multiplexed recording from adjacent cells with precisely defined alignment and separation using two independent nanoFET probes. The signal-to-noise ratio of our single nanoFET probe (∼100) is comparable to or smaller than those of vertical nanowire arrays (∼100 (ref. 8) and ∼590 (ref. 7)), although the effective areas of these passive nanoprobes are >50 times larger than our nanoFET probes. Hence, a direct comparison of signal and noise between these experiments is difficult to make because the signal and bandwidth are substantially degraded for these other devices when reduced to the same size as our current nanoFET probes.
Although future studies are needed to extend the performance and biochemical functionality of our free-standing nanowire probes, we believe this work opens up a number of interesting directions. First, our general approach for fabricating free-standing probes could be applied to other nanoelectronic building blocks that have been used in a chip-based format11,12,13,26. For example, the use of U-shape kinked nanowire structures would yield ultrasmall nanoFET detectors with a very high aspect ratio26, and thus enhance capabilities for specific targeting, multiplexed experiments and deep tissue/cell insertion and detection with subcellular resolution. Second, the small detector size and absence of ion exchange for our nanowire probes could facilitate studies of high-input impedance cells, such as cystic artery27, fibroblasts28 and glial cells29. There are also areas in which the nanoFET probes are currently limited compared to patch-clamp technology, including the capability to stimulate APs and deliver molecular and/or macromolecular reagents. Last, we believe that our approach could be scaled up in the future to make these novel 3D nanoelectronic probes accessible to a broad range of users in electrophysiology, bioelectronics and related fields.
Kinked silicon nanowires with nanoFETs encoded near the kink were synthesized using a gold nanocluster catalysed vapour–liquid–solid growth method as described previously15. Probe fabrication was carried out on Si substrates (Nova Electronic Materials) with 600 nm SiO2 coated with 100 nm Ni, which served as relief layer, and prebaked (65 °C for two minutes) SU8 2000.5 photoresist. Kinked nanowires were deposited on the desired region from ethanol dispersion using a micropipette, and then EBL was used to define the nanowire end of the probe and PL used to pattern the remainder of the 4.5 mm long SU8 probe body, which also served as the lower passivation of metal interconnects. Subsequently, a combination of EBL and PL steps was used to define the metal connections that scale from the kinked nanowire arms to the millimetre end of the probe, where Cr/Pd/Cr (1.5/120/60 nm) was used for contacts to the nanowire, and Cr/Au (5/200 nm) for the rest of the interconnections. Top SU8 passivation/structural layers were defined using a 500 nm thick SU8 2000.5 for the tip region covering the metal contacts, and a 50 µm thick low-stress SU8 layer (GLM 2060, Gersteltec) for the majority of the probe body, which can increase the overall mechanical strength. The kinked nanowire end of the probe was protected with 300 and 500 nm thick layers of LOR 3A and S1805 (MicroChem) defined by PL. After etching the Ni layer (40% FeCl3:39% HCl:H2O = 1:1:20), the floating probe structure was removed using tweezers and glued to the printed circuit board connector portion of the probe body mounted in the 3D manipulator. The protective cap was removed immediately prior to measurements following ultraviolet exposure, and measurements were made in a similar manner to those of previous chip-based device studies10,11. Additional fabrication details, as well as cell-culture and measurement protocols, are described in the Supplementary Information.
C.M.L. acknowledges support of this work by a National Institutes of Health Director's Pioneer Award (5DP1OD003900), National Basic Research Program of China (2013CB934103), and International Science & Technology Corporation Program of China (2013DFA50840).