Single cell in-vivo carbon nanotube device with multimodal sensingpotentialAlexandra Scavelli, Abhishek Gottipati, Everett Comfort, Sabarinath Jayaseelan, Thomas Murray et al. Citation: AIP Advances 3, 032122 (2013); doi: 10.1063/1.4795408 View online: http://dx.doi.org/10.1063/1.4795408 View Table of Contents: http://aipadvances.aip.org/resource/1/AAIDBI/v3/i3 Published by the AIP Publishing LLC. Additional information on AIP AdvancesJournal Homepage: http://aipadvances.aip.org Journal Information: http://aipadvances.aip.org/about/journal Top downloads: http://aipadvances.aip.org/features/most_downloaded Information for Authors: http://aipadvances.aip.org/authors

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AIP ADVANCES 3, 032122 (2013)

Single cell in-vivo carbon nanotube device with multimodalsensing potential

Alexandra Scavelli, Abhishek Gottipati, Everett Comfort, SabarinathJayaseelan, Thomas Murray, Michael Rizzolo, Scott Tenenbaum,a

and Ji Ung Leeb

College of Nanoscale Science and Engineering, University at Albany – State Universityof New York, Albany, NY 12203, USA

(Received 17 December 2012; accepted 1 March 2013; published online 8 March 2013)

Single walled carbon nanotube (SWNT) field effect transistors (NTFETs) are quicklybecoming the foundation for bioelectronic sensors. We describe a multimodal NTFETdevice that could be used as a real time single cell biosensor with the potential forchemical, optical and electrical sensing capabilities. This device utilizes the naturalmovement of a cell through the trench of a NTFET to provide a working cell-SWNT interaction where the nanotube is suspended. The use of individual suspendedSWNTs in lieu of non-suspended SWNTs in our device provides the basis for anin vivo NTFET multimodal single cell biosensor. Copyright 2013 Author(s). Thisarticle is distributed under a Creative Commons Attribution 3.0 Unported License.[http://dx.doi.org/10.1063/1.4795408]

Single-walled carbon nanotubes (SWNTs) have shown great potential for nanobiotechnologyapplications. In particular, SWNTs make optimal biosensors due to their specific and unique physicalcharacteristics.1 Their large length to diameter ratios provide a large surface area to volume ratio2

that can be utilized for both covalent3 and non covalent4 surface functionalization. The small sizesof SWNTs also play a significant role in their potential for biosensing applications. With a diameterof about 1 nanometer,5 SWNTs are size compatible with many biological entities of interest, suchas DNA,6 viruses,7 and proteins.8 The hollow nature of SWNTs also provides an opportunity fordifferent inner and outer surface functionalization9 for in vivo applications.10 Using their electronicand optical properties, a multimodal biosensor that combines electrical, chemical, and optical signalsis possible. Here, we demonstrate steps to incorporate a single SWNT inside a living cell in a devicethat can provide the potential for multimodal sensing capabilities.

Chemical and biological SWNT sensors are advantageous due to their high sensitivity to theenvironment and quick response times. As shown by Kong et al.11 single walled carbon nanotubefield effect transistors (NTFETs) operate with a sensitivity of up to three orders of magnitude andgas molecules, such as ammonia and nitrogen oxide, can be electrically detected within seconds.NTFETs can also detect electrical changes in a liquid environment.12 SWNTs retain a highlysensitive sensing capability within a biological environment, such as salt water, as charged moleculesnear the SWNT will act as an effective gate.13 To decrease the amount of nonspecific binding andincrease biocompatibility,14 individual sidewall defects15 can be created so that SWNTs can be point-functionalized to monitor single-molecule reactions.16 For example, individual SWNT biosensorshave been shown17, 18 to measure enzymatic activity and DNA-hybridization in real time using thistechnique.14 Past studies4 have had success with monitoring single-molecule dynamics throughnon-covalent functionalization, such as detecting the open and closed conformations of lysozymewith NTFETs in real time.19 SWNTs also show excellent photoluminescence properties and arephotostable in live cells.20 Furthermore, electrically induced luminescence21 from a single nanotube

aElectronic mail: stenenbaum@albany.edubElectronic mail: jlee1@albany.edu

2158-3226/2013/3(3)/032122/7 C© Author(s) 20133, 032122-1

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032122-2 Scavelli et al. AIP Advances 3, 032122 (2013)

FIG. 1. Schematic of the suspended SWNT device. The M4A4 cell travels into the trench that the suspended SWNT spans,resulting in partial insertion of the nanotube’s suspended portion into the cell, with the nanotube remaining functional.

can be achieved when it is fabricated as a p-n junction diode.22 Thus, if one were to incorporatesuch a device inside a cell, a pin-point location of the SWNT can be obtained that provides real-timecorrelation between signal and chemical reactions within the cell. Here, we have taken steps todemonstrate such a sensor inside a cell.

Several studies23 have modified NTFETs to increase their functions as biosensors. Most ofthese modified NTFETs involve non-suspended SWNTs,24–27 or a network of SWNTs.28 Herein,we propose a novel single cell in vivo FET biosensor based on individual suspended SWNTs in adevice that can function both as a FET and a p-n junction diode. Advantages of suspended SWNTsinclude a greater availability of surface area, which increases the sensitivity1 of the device, and theability of the cell to incorporate the nanotube internally while preserving the electrical connectivityto the carbon nanotube. Figure 1 schematically depicts our device structure. The trench (.3-1.5 μm)where the SWNT is suspended allows for a natural uptake of the SWNT by the cell, as opposedto more invasive procedures where nanotubes are used as probes that forcibly penetrate the cellmembrane.29, 30 Here, we demonstrate the electrical continuity of the carbon nanotube before andafter it is naturally taken up by the cell, demonstrating an in vivo biological sensor at the singlecellular level.

Our parallel scheme for device fabrication produces several hundred devices on a die, with thefinal device yield determined by our nanotube growth. The first step of the process is to thermallygrow a 100 nm thick layer of SiO2 on a silicon substrate. Next, we deposit 100 nm of polysilicon,then implant the layer with phosphorous and anneal to activate the dopants, making the layer highlyconductive. The polysilicon is then patterned using photolithography and reactive ion etching (RIE)techniques. The patterning forms the split gates shown in Fig. 1 that are used to reconfigure the deviceas either a FET or p-n diode. When both gates are equally biased or when the substrate is biasedthe device functions as a back gated FET. When the split gates are biased with opposite voltages,a p-n junction diode is formed along a nanotube.22 After patterning the split gates, a layer of SiO2

is deposited using chemical vapor deposition and planarized to a final thickness of 100 nm abovethe polysilicon. Contacts to the bondpads are created by etching through the SiO2 to contact theburied polysilicon. After which, a 50 nm layer of titanium nitride (TiN) is deposited and patterned,serving as the contact metal to the carbon nanotube. Using a photolithography and reactive ionetch process, a trench in the center of the device is created approximately 300 nm deep, which willsuspend the carbon nanotube. Carbon nanotubes spanning up to 3 μm over the trench are suspendedin our devices. A layer of PMMA is patterned, and catalyst for growing nanotubes is spun onto thewafer. The PMMA is lifted off in acetone, creating lithographically defined catalyst regions. Finally,the carbon nanotubes are grown using a catalytic chemical vapor deposition process.31 Over 7% of

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the devices have nanotubes across the trench that are semiconducting. Of these, many will have acell that has migrated into the trench after the die has been incubated with mammalian cells usingstandard cell-culture techniques. Specifically, thousands of devices were initially tested and thereare about 400 devices per die. On average, over a dozen working devices resulted with cell growthin the trench per die and about half of these were semiconducting.

The trench is a critical component to allow the natural growth and subsequent horizontal uptake(insertion) of the nanotube into a cell. We have noted that cells prefer to move through the trenchrather than remain on the top SiO2 surface. When a nanotube is partially suspended as shown inFig. 1, the growth of a cell along the trench forces the cell to encounter and subsequently engulfthe nanotube, resulting in partial cellular inclusion of the nanotube. This is demonstrated by thepreserved integrity of the nanotube after its uptake by the cell. Remarkably, the small diameter of thehorizontal nanotube does not dramatically disturb the integrity of the cell membrane or the viabilityof the cell, resulting in a probe that is situated internal to the cell but remains externally contactedelectrically. Our results also demonstrate a viable method that does not require carbon nanotubes tobe modified with phospholipid bilayers in order to penetrate the cell membrane, in comparison tostudies30 where FET functionalization was required for uptake by the cell.

The individual carbon nanotubes grown can be either metallic or semiconducting, which wedetermine by measuring the drain-source current, Ids, as a function of the gate voltage, Vg. Afterdetermining each device characteristic, the devices were sterilized by rinsing in a 70% ethanol(C2H6O) solution prior to addition of M4A4 human breast cancer derived MDA-MB-435 cells.This ethanol rinse disinfected the nanotubes without affecting the yield of the devices. After thecell growth and fixation, 70% of carbon nanotube devices were still functional, as determinedfrom the transfer curves that showed similar behavior to the characteristics before the cell growth.The M4A4 cell line was chosen for its short cell division rate, with a doubling rate of approx-imately 30 hours. The cells were cultured and grown in an ATCC-formulated Dulbecco’s Mod-ified Eagle’s Medium supplemented with 10% fetal bovine serum over a period of two to threedays.

After the devices were prepared for cell growth, they were placed in tissue culture platescontaining growth medium and ∼1 million cells. Following overnight incubation, the M4A4 cellson the devices were fixed with a solution of 3% glutaraldehyde in .1 M phosphate buffer (pH7.4) containing .1 M sucrose for 2 hours. After fixing the cells, the devices were left to airdry and were re-characterized by taking electrical measurements. After identifying the work-ing devices, visual inspection was required to identify devices with cells localized within thetrench.

Electrical data, in conjunction with physical analysis, confirmed the picture of the nanotubeand cell as shown in Figure 1. More specifically, we use current-voltage (I-V) curves taken beforeand after cell growth to confirm that the electrical behavior of the SWNT remains unchanged,scanning electron microscopy (SEM) analysis to determine the location of the CNT inside the cell,and focused ion beam (FIB) cross-sections to confirm that the cell does indeed enter the trench.Figure 2(a) and 2(b) shows optical images of a working device with a significant part of the cell,including the cytoplasmic region, within the trench. Cellular uptake of the SWNT did not affect thefunctionality of the device, as I-V curves were similar before and after cell growth with the exceptionof an increase in resistance of the on-current after cell growth (Figure 2(c)). Previous studies suggestthat the increase of resistance could be a result of a charge transfer reaction that donates electronsto the nanotube.32 We note that a device with a cell and no carbon nanotube is electrically similarto the background noise of a device. The large on/off ration seen in the I-V curve confirms that thenanotube is still semiconducting and functioning. But, most importantly, the ambipolar characterwhich is required to bias the device as a p-n junction is preserved.

After confirming the electrical continuity of the nanotube, SEM analysis was conducted to showthat the nanotube was intact and within the cell. To conclude that the CNT is within the cell, insteadof lying on top of the cell, SEM images were taken at 1, 5, and 7 kilovolts (kV). At 1 kV (Figure 3(a)),the nanotube is not visible within the indicated area, showing that electron beam only penetratesthe surface of the cell and does not reach the nanotube, which is located deeper within the cell. At5 kV (Figure 3(b)) in the indicated region, the nanotube is visible within the cell, showing that the

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FIG. 2. (a) Optical picture of the device cell growth. (b) Optical picture demonstrating that the cell has migrated up thetrench, partially internalizing the suspended portion of the SWNT. (c) Before and after cell growth IV curves of a singledevice, showing that the device is still functional after cell growth, and the on-current resistance increases.

CNT is deeper within the cell than the 1 kV energy could penetrate. The intact CNT is fully visibleat an energy of 7 kV (Figure 3(c)), at a depth within the cell that the 7 kV beam can penetrate. Thesuspended SWNT is shown to have made contact at the source and drain in Figure 3(a), which wastaken at 1.00 kV, while Figure 3(c) shows that the nanotube fully spans the trench between the metalcontacts while embedded within the cell. We estimated the depths of the electron beam penetration,and therefore the size of the cell and the depth of the nanotube within the cell, using the equation33

x(μm) = .1E1.50 /ρ. In the equation, E0 is the accelerating voltage (kV), ρ is the density of the sample

(g/cm3), and x is the depth of electron penetration (μm). The density of the cell was estimated afteradjusting the density of an adipose cell34 to reflect a density decrease of 2.5% after cell fixation.35

At 1, 5, and 7 kilovolts (kV), the estimated electron penetration depths are .114 μm, 1.27 μm, and

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FIG. 3. SEM false color image of the fixed M4A4 cell (yellow) and the suspended SWNT at (a) 1 kV, (b) 5 kV, and (c) 7 k,showing the location of the carbon nanotube within the cell at different penetration depths.

2.11 μm, respectively. These estimates are consistent with Figure 4(d), which shows that the cell isapproximately 2 μm tall at its highest point.

The depth of the cell within the trench, and therefore the interaction of the cell and the carbonnanotube, is further supported by FIB cross section images shown in Figure 4(a)–4(d). After identi-fying the location of the nanotube on a different device, a FIB cut is made at the precise location ofthe cell where the nanotube is suspended.

For FIB cross-sections, samples were mounted on an aluminum stub using silver paint and RFsputter coated with Au/Pd. All FIB/SEM was done at high vacuum (<5 × 10-5 Torr) on an FEI NovananoLab 600 Dualbeam FIB/SEM. The area of interest was coated with electron beam depositedplatinum to protect the underlying structure during FIB milling. FIB cuts were carried out using a30 kV, 0.28 nA Ga+ ion beam. The cuts used a cleaning cross-section pattern and a Si-ccs applicationfile with an overlap of 50%. Secondary electron images were acquired after each slice. Images weretaken at 2 kV with a 0.21 nA beam at a working distance of 5.1 mm using the In-lens detector.

Although we are not able to visualize the nanotube after the FIB cut, the topology of the cellwithin the trench makes it obvious that the nanotube is in the cell, as pictured in Fig. 1. As seenin Figure 4(c) and 4(d), the cell completely enters the trench, confirming that our carbon nanotubeis inside the cell as the nanotube is known to be present in the trench, based on our electricalcharacterization in Figure 3. The volume of the cell that resides in the trench and the topology ofthe cell in the trench are a function of the width of the trench. In a relatively wide trench, as shownin Fig. 4(c), the cell resides fully below the top level of the trench. In narrower devices, like the one

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FIG. 4. SEM and FIB cross section images showing the cell within the trench. SEM images depicting the milling regionbefore (a) and after (b) the device was FIB cross sectioned. Ion beam images of a device with a 1.5 μm trench (c) and adevice with a.3 μm trench (d) show that the cell completely enters the trench where the carbon nanotube is present.

examined in Fig. 4(d), a large portion of the cell, up to several microns, can reside both in and abovethe trench, consistent with the penetration depth estimated from the beam energies.

In summary, we demonstrate a SWNT biosensor with multimodal sensing capabilities at thesingle cell level. This is accomplished by having a nanotube, in a device that can be configured as aFET or a diode, thread through a cell to allow in vivo sensing capabilities. The uptake of the SWNTwas accomplished by partially suspending the SWNT and was confirmed through electrical testing,SEM analysis, and FIB imaging. Our multimodal biosensor should provide electrical, chemical, and

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optical sensing capabilities, and may provide new opportunities for exploring cellular processes indepth.

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