We present for the first time a functional planar SiN-nanoneedle system for intracellular mass transport and in vivo electrophysiological measurements on-chip. Though several micro- or nanoneedles for cell research have been described in literature, no needles of this small size equipped with nanosized inner channels or electrodes have been reported. A propidium iodide assay verifies the excellent penetration performance of the nanoneedles with diminished leakage from the cell after insertion and release of the needle from HL60- cells. Hollow needles connected to on-chip sub-picoliter electrochemical dosing systems are in development

Andersson, Helene

Emmelkamp, Jurjen

Gardeniers, Johannes G.E.

van den Berg, Albert

English

University of Twente Research Information

PLANAR NANONEEDLES ON-CHIP FORINTRACELLULAR MEASUREMENTSJ. Emmelkamp1, J.G.E. Gardeniers1, H. Andersson1,2, A. van den Berg11MESA+ Institute, Lab-on-a-Chip group, University of Twente, The Netherlands2Royal Institute of Technology, Dep. Of Signals, Sensors and Systems, SwedenABSTRACTWe present for the first time a functional planar SiN-nanoneedle system for intracellularmass transport and in vivo electrophysiological measurements on-chip. Though severalmicro- or nanoneedles for cell research have been described in literature, no needles of thissmall size equipped with nanosized inner channels or electrodes have been reported. Apropidium iodide assay verifies the excellent penetration performance of the nanoneedleswith diminished leakage from the cell after insertion and release of the needle from HL60-cells. Hollow needles connected to on-chip sub-picoliter electrochemical dosing systemsare in development.Keywords: Nanoneedles, intracellular, micro-machining, nanofluidics1. INTRODUCTIONFor intracellular research several techniques are used (e.g. patch clamping,electroporation), but mechanical hollow needles are superior for their simplicity andefficiency. In contrast with conventional pulled glass micropipettes the presented SiN-planar needles can be made by micromachine techniques. Micromachined transdermal drugdelivery microneedles, presented at µTAS2003 by the groups of Horiike [1] and ours [2],are shaped as vertically standing pillars and can have microchannels inside, but due to theused dimensions (50 µm to 1 mm) these needles are not very suitable for intracellular use,and the pillar shape is a disadvantage for on-chip applications. Recently Obataya et al.presented nanoneedles that gave excellent results on penetration of the cell membranewithout killing the cell [3], but their nanoneedles were not equipped with inner channels orelectrodes.2. FABRICATIONThree different needles are produced: hollow needles (figure 1), needles with electrodesof gold, silver or platinum (figure 2) or only plane (figure 3). In the hollow and electrodeneedles the channels or electrodes are encapsulated between a LPCVD-nitride bottom layer(300 nm) and a PECVD-nitride top layer (300 nm). Channel opening or electrode tip isplaced at the needle tip. The plane needle only has the LPCVD layer. The needles areetched by reactive ion etching and the channels are made by sacrificial layer technique. Asacrificial chromium line is etched by ion beam etching, after deposition of the top layer thesacrificial material is removed. The electrodes are formed by wet etching. The planarneedles are placed over a well, etched in silicon by KOH.9th International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 9-13, 2005, Boston, Massachusetts, USA400 0-9743611-1-9/µTAS2005/$20©2005TRF9th International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 9-13, 2005, Boston, Massachusetts, USA401Figure 1. SEM-picture of a hollownanoneedle. The channel is clearly visiblein the middle of the needle. Inner channeldimensions: 150 nm high, 2 µm wide.Needle dimensions: 0.7 µm high, 3 µm wideand 5 µm long. The needle is sharpened byfocused ion beam etching.Figure 2. SEM-picture of an electrodeneedle. The electrode tip is 2 µm long, 2µm wide and 100 nm high. Needledimensions as in Figure 1.Figure 3. Three plane nanoneedles, markedwith 1, 2, and 3. Below the nanoneedles, inthe silicon bottom of the well, dicing linesare visible, produced during focused ionbeam cutting of the sharp needle shape.Needle dimensions: 260 nm high, 3 µm wideand 10 µm long.Figure 4. Microscopic picture of a HL60-cell punctured by a nanoneedle. The cellis positioned in the middle of the whitesquare; the needle tip is the white tip inthe top of the white square. In the lowerleft corner the external micropipette isshown.9th International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 9-13, 2005, Boston, Massachusetts, USA4023. RESULTS AND DISCUSSIONIn figure 4 a HL60-cell is placed at the needle tip by an external micropipette, marked bythe white square. After removing the cells from the nanoneedle a solution of propidiumiodide was added to stain non-viable cells. After puncturing the cell membrane many cellswere still viable. During these preliminary measurements no cell leakage was observed.An electrochemical sub-picoliter dosing system is currently in development. This dosingsystem will be capable to dose volumes small enough not to rupture the cell membranewhile injecting drugs, chemicals or biological samples into a normal sized cell and largeenough to get a chemical analysis measurement from the cell contents after an extraction.4. CONCLUSIONSWe demonstrated a nanoneedle suitable for biomedical cell research. Injection into andextraction from viable HL60 cells will be realized by means of integration of thedemonstrated hollow nanoneedles with the sub-picoliter dosing system. In vivoelectrophysiological measurements will be performed using the electrode needles.ACKNOWLEDGEMENTSFinancial support from STW (project TMM 6016, “NanoSCAN”) and valuable work byRamona Bouwman, Jan van Nieuwkasteele, Regina Luttge and Willem Engel is gratefullyacknowledged.REFERENCES[1] S. Momese, T. Fukasawa, H. Takasu and Y. Horiike, Painless Si needle array chipcollecting blood from capillary vessel and combined centrifugal separation chip,µTAS2003, p. 211-214.[2] R. Luttge, J.G.E. Gardeniers, E.X. Vrouwe and A. van den Berg, Microneedle arrayinterface to CE on chip, µTAS2003, p. 511-514.[3] I. Obataya, C. Nakamura, S.W. Han, N. Nakamura and J. Miyake, Nanoscaleoperation of a living cell using an atomic force microscope with a nanoneedle, Nanoletters,5, 1, p. 27-30 (2005).Contact address: Jurjen Emmelkamp, MESA+ Institute, University of Twente, BIOS-group,P.O. Box 217, 7500 AE, Enschede, The Netherlands.Tel. +31-53-489-3944; e-mail: j.emmelkamp@utwente.nl

Planar Nanoneedles on-chip for intracellular measurements

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