It is shown that buried graphitic channels fabricated in monocrystalline diamond by selective damage induced by focused MeV ions, can be considered an effective alternative to the commonly used carbon-fibers to detect the catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis. Quantal secretory responses have been measured from stimulated chromaffin cells, which were positioned on the graphitic microelectrode, polarized to +800 mV. Sequences of amperometric spikes started after cell stimulation with the KCl solution, with amplitudes well above the background noise within the range of 8–180 pA and comparable with signals obtained by conventional carbon fiber electrodes

Picollo, F.

English

Scientific Open-access Literature Archive and Repository

DOI 10.1393/ncc/i2013-11550-2Communications: SIF Congress 2012IL NUOVO CIMENTO Vol. 36 C, N. 4 Luglio-Agosto 2013Amperometric detection of quantal catecholamine secretionfrom individual cells by an ion beam microfabricatedsingle crystalline diamond biosensorF. Picollo(∗)Physics Department - University of Torino, Torino, ItalyNIS Centre of Excellence - University of Torino, Torino, ItalyINFN, Sezione di. Torino, Torino, Italy andConsorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM)Sezione di Torino, Torino, Italyricevuto il 31 Dicembre 2012Summary. — It is shown that buried graphitic channels fabricated in monocrys-talline diamond by selective damage induced by focused MeV ions, can be consid-ered an effective alternative to the commonly used carbon-fibers to detect the cate-cholamine release from cells as individual secretory granules discharge their contentsduring the process of quantal exocytosis. Quantal secretory responses have beenmeasured from stimulated chromaffin cells, which were positioned on the graphiticmicroelectrode, polarized to +800mV. Sequences of amperometric spikes startedafter cell stimulation with the KCl solution, with amplitudes well above the back-ground noise within the range of 8–180 pA and comparable with signals obtained byconventional carbon fiber electrodes.PACS 85.40.Hp – Lithography, masks and pattern transfer.PACS 81.05.uj – Diamond/nanocarbon composites.PACS 87.85.D- – Applied neuroscience.1. – IntroductionFollowing our previous studies on the formation of conductive graphitic microchan-nels in single-crystal diamond by means of direct lithography with a scanning ion mi-crobeam [1-5], it is reported on the fabrication and preliminary testing of a cellularbiosensor with the above-mentioned technique.The so-called “Lab-on-a-chip” devices are highly demanded in modern biotechnologyto cultivate living cells for long periods while inducing and revealing a broad range ofelectrical biosignals, such as the quantal release of bioactive molecules from neurons andneuroendocrine cells.(∗) E-mail: picollo@to.infn.itc© Societa` Italiana di Fisica 153154 F. PICOLLOAn integrated diamond-based device can effectively address many open issues in theexisting cell-sensing research which to different extents are not met by conventionalbiomaterials (silicon [6], gold [7, 8], platinum [9, 10], indium-doped tin oxide [11-13],diamond-like carbon [14-16], carbon nanotubes [17] and conductive polymers [18]), suchas robustness and reproducibility in performance over repeated biosensing cycles, bio-compatibility and long-term stability for in vivo measurements and high transparencyfor optical interfacing.Here it is reported on the MeV-ion-beam fabrication process of a diamond cellularsubstrate with graphitic electrodes, and on the results of its preliminary characterizationfor the detection of exocytosis activity from chromaffin cells. The results obtained withthe diamond biosensor are also compared with conventional electrodes such as carbonmicrofibers.Carbon fiber microelectrodes (CFEs) are employed since few decades to detect theexocytotic activity of single excitable cells with amperometric techniques and providesignificant information on key mechanisms such as the formation of the fusion pore andthe kinetics of single secretory events in real time [19-22].2. – Device microfabricationIn the present work an artificial single-crystal diamond sample produced by SumitomoElectric by means of “high pressure high temperature” (HPHT) technique was employed.Sample size is 3×3×1.5mm3 and the crystal is classified as type Ib, i.e. its substitutionalnitrogen concentration is comprised between 10 and 100 ppm.The sample was implanted at the ion microbeam line of the AN2000 accelerator ofthe Legnaro National Laboratories with a scanning beam of 1.8MeV He+ ions at typicalfluences of ∼ 5·1017 cm−2. Ion beam size was 10μm, while beam currents were comprisedbetween 5 nA and 8 nA, thus ensuring typical implantation times of 50 minutes. Thesample was metal-coated to avoid surface charging and fluence was accurately monitoredwith an electrometer connected to the sample chamber, which is electrically insulatedfrom the rest of the beamline, thus acting effectively as a Faraday cup.As reported in previous works [1-5], high-fluence MeV ion implantation determinesthe formation of a sub-superficial amorphized layer in correspondence of the ion end-of-range. In the present work, ion implantation led to the formation of a ∼ 300 nm thickamorphous layer at a depth of 3.2μm below the sample surface. The employment ofvariable-thickness masks defined on the sample surface by means of non-contact metalevaporation allowed the gradual modulation of the penetration depth of the MeV ions,thus ensuring the emergence of the buried layers at the sample surface at specific loca-tions, as schematically shown in fig. 1.After ion implantation, the sample was annealed in vacuum at a temperature of1100 ◦C for 2 hours. The process resulted in the conversion of the amorphized layer toa graphitic phase, while the surrounding regions which were damaged below a criticalthreshold reconverted to the pristine diamond form. One of the electrically conductivegraphitic microchannels was subsequently connected to the acquisition electronic chainby means of a standard metal contact, while the other emerging end defined the locationat the sample surface where biochemical sensing was performed, as schematically shownin fig. 2.It is worth underlying that the micro-electrode is employed to monitor the activityof a living cell, which during the in vitro measurements is immersed in a physiologicalsolution: for this reason buried connections embedded in the insulating diamond matrixAMPEROMETRIC DETECTION OF QUANTAL CATECHOLAMINE SECRETION ETC. 155Fig. 1. – Schematics of the variable-thickness process allowing the definition of the buried amor-phous layer at variable depth.are demanded, and the metal macro-contacts are shielded with a biocompatible insulatingmask in “sylgard” polymer.Before proceeding with the functional tests reported in the following section, two-points electrical characterization (see fig. 3) was performed to check that i) the buriedelectrical channels get in electrical contact with the sample surface at their endpoints andii) the electrical resistivity of the microchannels is compatible with that of polycrystallinegraphite (i.e. ∼ 3 · 10−3 Ωcm).Fig. 2. – Top: Lateral view schematics of the diamond device incorporating the graphitic mi-crochannel. Bottom: top view micrograph of the microelectrode at the endpoint of the graphiticmicrochannel.156 F. PICOLLOFig. 3. – Current-voltage characteristic of buried conductive channel.3. – Exocytosis detection from chromaffin cellsThe exocytosis of neurotransmitters is the biological process taken under exam andhas been studied by means of detection of adrenaline secretion from the chromaffin cellswith the diamond-base biosensor.Those cells are located into the medulla part of the adrenal gland, are characterizedby the presence inside them of granules. Those granules have size ranged from 150 to350 nm and contain high concentration of catecholamines as adrenaline and noradrenaline(0.5–1M).Stress conditions induce the medulla gland to secrete catecholamines in order to stim-ulate target organs (i.e. heart frequency increase, bronchus dilatation, etc.).The chromaffin cells can be studied as model for neuron secretion since those areelectrically excitable cells, have an exocytic apparatus analogous of neurons one andsecrete the same family of neurotransmitters (catecholamines). Moreover, chromaffincells have large dimension (10–20μm) then neuron’s synapses and are easily cultivableand isolable.The responsiveness of the device towards catecholamines (adrenaline, noradrenaline)was preliminarily tested by means of cyclic voltammetry measurements.As shown in fig. 4, when immersed in a saline solution containing 10mM adrenaline,the device can detect the oxidation of the adrenaline molecules at the electrode when thevoltage reaches +0.8V, as opposed to the case when a standard saline solution (in mM:128 NaCl, 2 MgCl2, 10 glucose, 10 HEPES, 10 CaCl2, 4 KCl) is employed.Following these preliminary tests, the device was tested by positioning an isolatedchromaffin cell in correspondence of the active region, while keeping the whole system inphysiological solution.The micro-electrode was polarized at the fixed voltage corresponding to the oxidationof the adrenaline (i.e. 0.8V, as reported in fig. 4), in order to detect, via amperometry,the quantal release occurring during exocytosis. Perfusion of KCl-enriched solution wasemployed to stimulate the adrenaline secretion from chromaffin cells. As reported infig. 5, the device is able to detect with high signal-to-noise ratio the amperometric spikesof 3–22 pA corresponding to the quantal release of catecholamines.AMPEROMETRIC DETECTION OF QUANTAL CATECHOLAMINE SECRETION ETC. 157Fig. 4. – Cyclic voltammetry tests on saline and adrenaline-containing solutions. The adrenalineoxidation feature at V = 0.8V is clearly visible.Systematic data analysis of amperometric spikes was performed by means of “Igor”macros, as previously described in [23]. The amplitude of most visible amperometricspikes were well above the background noise (5 pA peak-to-peak) and varied from 8pAto 180 pA. The background noise of graphitic microelectrode has the same amplitude ofCFE one (data not reported).Fig. 5. – Time course of amperometric signals detected from a single chromaffin cell located incorrespondence of the active region of the device: the spikes (zoom in the inset) correspond tosingle exocytotic events.158 F. PICOLLOFig. 6. – Histograms of characteristic parameters of spikes. Each graph shows the comparison ofthe results obtained with carbon fibre electrode and micrographitic channels. The representedspikes’s parameters are: Imax maximum of current amplitude, m slope of gradient, Q integral ofthe peak (total charge measured), Q1/3 cube root of Q, tp time needed to reach the maximumof the peak, t1/2 full width at half maximum of the peak.Information of exocytosis process are obtained by the characteristic parameters ofamperometric spikes.The parameters analyzed for our measurements are summarized below and repre-sented in the fig. 6:– Imax: maximum of current amplitude due to adrenaline oxidation;– Q: integral of the peak which is the total charge of one granule;– Q1/3: cube root of Q, this parameter is proportional to the granules’ radius;– m: slope of gradient gives information concerning the evolution of fusion pore (aper-ture that allows the release of neurotransmitters from the granule to the outside ofthe cell membrane);– tp: time needed to reach the maximum of the peak, it indicates the time in whichthe granule releases all the adrenaline;– t1/2: full width at half maximum of the peak.As indicated in fig. 6, amperometric spikes recorded by the graphitic microelectrode(n = 94 spikes) are comparable with signals obtained by conventional CFEs. Thisis particularly clear comparing the more interesting biological parameters as the meancurrent amplitude (26±4 pA and 33±6 pA values measured with graphitic microelectrodeAMPEROMETRIC DETECTION OF QUANTAL CATECHOLAMINE SECRETION ETC. 159and CFE, respectively) and the mean collected charge (0.28 ± 0.04 pC versus 0.35 ±0.07 pC measured with graphitic microelectrode and CFE, respectively). The discrepancyobserved for the tp parameter is probably due to difference on cells’ stimulation (i.e. non-equal volumes of perfused KCl-enriched solution) that justify also a large error bar forCFE measurement.4. – ConclusionsBuried conductive micro-channels were fabricated by means of direct writing with ascanning MeV ion-beam, taking advantage of the possibility offered by a high-energyion probe to locally amorphize and subsequently graphitize the diamond structure. Invitro detection of quantal neurotransmitter release from single excitable cells kept in aphysiological solution was performed.It is worth noting that the performance (amperometric sensitivity, signal-to-noise ra-tio, time resolution) of the prototypical diamond-based device presented in this workcompare well not only with standard CFEs (see fig. 6), but also with other well de-veloped technologies at the state of the art, such as devices based on indium tin oxide(ITO) conductive glass [12, 13, 17], noble metals (Au, Pt, . . . ) [8, 15] and boron-dopednanocrystalline diamond (B:NCD) [23]. In comparison with devices based on CFEs,ITO and noble metals, our device offers the advantages of an all-carbon architecturewhich is advantageous in terms of chemical stability and biocompatibility, and a broaderelectrochemical window. In conclusion, these results open promising perspectives forthe realization of all-carbon multielectrode miniaturized devices in artificial diamond (amaterial which is becoming available with increasing crystal quality at ever-decreasingcosts [24, 25] in which full advantage of the robustness, chemical stability, biocompat-ibility and transparency can be exploited to obtain multiparametric signals detectionfrom cell networks. This approach has the potential to drastically accelerate data col-lection from large ensembles of cells, leaves the experimental sample under physiologicalconditions. In addition, it is non-invasive and allows repeated measurements over time.Furthermore, geometry and transparency of the structures allows multi-techniques mea-surements, since other probes (i.e. patch-clamp pipettes) can be easily adapted.∗ ∗ ∗This work was supported by: “FIRB - Futuro in Ricerca 2010” project (CUP code:D11J11000450001), funded by the Italian Ministry of Education, University and Research(MIUR); “Micro.Di.Bi.” project, funded by the “BioPMed” scientific pole of RegionePiemonte; “Linea 1A ORTO11RRT5” projects of the University of Torino, funded by“Compagnia di San Paolo”.REFERENCES[1] Olivero P., Amato G., Bellotti F., Budnyk O., Colombo E., Jaksˇic´ M., LoGiudice A., Manfredotti C., Pastuovic´ Zˇ., Picollo F., Skukan V., Vannoni M.and Vittone E., Diamond Relat. Mater., 18 (2009) 870.[2] Olivero P., Amato G., Bellotti F., Borini V, Lo Giudice A., Picollo F. andVittone E., Eur. Phys. J. B, 75 (2) (2010) 127.[3] Picollo F., Olivero P., Bellotti F., Pastuovic´ Zˇ., Skukan N., Lo Giudice A.,Amato G., Jaksˇic´ M. and Vittone E., Diamond Relat. Mater., 19 (2010) 466.160 F. PICOLLO[4] Olivero P., Forneris J., Jaksˇic´ M., Pastuovic´ Zˇ., Picollo F., Skukan N. andVittone E., Nucl. Instrum. Methods B, 269 (2011) 2340.[5] Picollo F., Gatto Monticone D., Olivero P., Fairchild B. A., Rubanov S.,Prawer S. and Vittone E., New J. Phys., 14 (2012) 053011.[6] Chen P., Xu B., Tokranova N. B., Feng X., Castracane J. and Gillis K. D., Anal.Chem., 75 (2003) 518.[7] Cui H. F., Ye J. S., Chen Y., Chong S. C. and Sheu F. S., Anal. Chem., 78 (2006)6347.[8] Dittami G. M. and Rabbitt R. D., Lab Chip, 10 (2010) 30.[9] Dias A. F., Dernick G., Valero V., Yong M. G., James C. D., Craighead H. G.and Lindau M., Nanotechnology, 13 (2002) 285.[10] Hafez I., Kisler K., Berberian K., Dernick G., Valero V., Yong M. G.,Craighead H. G. and Lindau M., Proc. Natl. Acad. Sci. U.S.A., 102 (2005) 13879.[11] Sun X. and Gillis K. D., Anal. Chem., 78 (2006) 2521.[12] Chen X., Gao Y., Hossain M., Gangopadhyay S. and Gillis K. D., Lab Chip, 8(2008) 161.[13] Meunier A., Fulcrand R., Darchen F., Guille Collignon M., Lemaˆıtre F. andAmatore C., Biophys. Chem., 162 (2012) 14.[14] Parpura V., Anal. Chem., 77 (2005) 681.[15] Gao Y., Chen X., Gupta S., Gillis K. D. and Gangopdhyay S., Biomed. Microdevices,10 (2008) 623.[16] Sen A., Barizuddin S., Hossain M., Polo-Parada L., Gillis K. D. andGangopadhyay S., Biomaterials, 30 (2009) 1604.[17] Shi B. X., Wang Y., Zhang K., Lam T. L. and Chan H. L. W., Biosens. Bioelectron.,26 (2011) 2917.[18] Larsen S. T. and Taboryski R., Analyst, 137 (2012) 5057.[19] Chow R. H., von Ru¨den L. and Neher E., Nature, 356 (1992) 60.[20] Travis E. R. and Wightman R. M., Annu. Rev. Biophys. Biomol. Struct., 27 (1998) 77.[21] Wightman R. M., Science, 311 (2006) 1570.[22] Amatore C., Arbault S., Guille M. and Lemaˆıtre F., Chem. Rev., 108 (2008) 2585.[23] Carabelli V., Gosso S., Marcantoni A., Xu Y., Colombo E., Gao Z., VittoneE., Kohn E., Pasquarelli A. and Carbone E., Biosens. Bioelectr., 26 (2010) 92.[24] Butler J. E., Mankelevich Y. A., Cheesman A., Ma J. and Ashfold M. N. R., J.Phys. Cond. Matter, 21 (2009) 364201.[25] Teraji T., Phys. Status Solid. A, 203 (2006) 3324.

Amperometric detection of quantal catecholamine secretion

from individual cells by an ion beam microfabricated single crystalline diamond biosensor

http://eprints.bice.rm.cnr.it/18295/1/ncc10564.pdf

Amperometric detection of quantal catecholamine secretion from individual cells by an ion beam microfabricated single crystalline diamond biosensor

Abstract

Similar works

Full text

Available Versions

Scientific Open-access Literature Archive and Repository