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Simple and effective graphene laser processing for neuron patterning application.

Lorenzoni M, Brandi F, Dante S, Giugni A, Torre B - Sci Rep (2013)

Bottom Line: Primary embryonic hippocampal neurons were cultured on our substrate, demonstrating an ordered interconnected neuron pattern mimicking the pattern design.Surprisingly, the functionalization is more effective on the SLG, resulting in notably higher alignment for neuron adhesion and growth.Therefore the proposed technique should be considered a valuable candidate to realize a new generation of highly specialized biosensors.

View Article: PubMed Central - PubMed

Affiliation: Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy. matteo.lorenzoni@iit.it

ABSTRACT
A straightforward fabrication technique to obtain patterned substrates promoting ordered neuron growth is presented. Chemical vapor deposition (CVD) single layer graphene (SLG) was machined by means of single pulse UV laser ablation technique at the lowest effective laser fluence in order to minimize laser damage effects. Patterned substrates were then coated with poly-D-lysine by means of a simple immersion in solution. Primary embryonic hippocampal neurons were cultured on our substrate, demonstrating an ordered interconnected neuron pattern mimicking the pattern design. Surprisingly, the functionalization is more effective on the SLG, resulting in notably higher alignment for neuron adhesion and growth. Therefore the proposed technique should be considered a valuable candidate to realize a new generation of highly specialized biosensors.

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Scanning Kelvin probe force microscopy.Topography (a) and potential map of type 2 sample irradiated at (a, b) 0.12 J/cm2 and (c) 0.22 J/cm2; in (b) along a random horizontal line the ΔCP between pristine SLG and modified area is −98 ± 4 mV, while an increase of CPD is clearly visible all along the edges of the laser irradiation; between the blue arrows the increase is 50 ± 4 mV, compatible with a local folding of graphene layers; topography (a) cannot show any contrast. All images refer to a 20 × 40 μm2 area. In (d), plot of averaged CPD variation along X for three different irradiation powers: 0.09, 0.12, 0.22 J/cm2. Potential scale baselines (CPD SLG) have been set to zero. In (e) we plot two CPD profiles of the boundaries zones of scan (c), line 1 in black color is the left edge profile and line 2 in red color is the right edge profile as depicted in image (c).
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f6: Scanning Kelvin probe force microscopy.Topography (a) and potential map of type 2 sample irradiated at (a, b) 0.12 J/cm2 and (c) 0.22 J/cm2; in (b) along a random horizontal line the ΔCP between pristine SLG and modified area is −98 ± 4 mV, while an increase of CPD is clearly visible all along the edges of the laser irradiation; between the blue arrows the increase is 50 ± 4 mV, compatible with a local folding of graphene layers; topography (a) cannot show any contrast. All images refer to a 20 × 40 μm2 area. In (d), plot of averaged CPD variation along X for three different irradiation powers: 0.09, 0.12, 0.22 J/cm2. Potential scale baselines (CPD SLG) have been set to zero. In (e) we plot two CPD profiles of the boundaries zones of scan (c), line 1 in black color is the left edge profile and line 2 in red color is the right edge profile as depicted in image (c).

Mentions: Scanning Kelvin probe force microscopy measures local contact potential difference (CPD) between a conductive AFM tip and a sample. This potential difference is sensitive to local compositional and structural variations. The work function of graphene is similar to that of graphite (~4.6 eV)27 and shows a dependence on the number of layers, ranging from 4.51 eV for SLG to 4.58 eV for AB stacked HOPG28. Bi-layer and multilayer graphene should show an increase in work function of 50–100 mV due to its improved chemical stability27. Based on these considerations we performed AFM topography (Fig. 6a) and CPD scan (Fig. 6b–c) over irradiated areas. The work function of one reference tip (Φtip = 4.93 ± 0.05 eV) was calibrated on freshly cleaved highly oriented pyrolytic graphite (HOPG), as previously reported in literature29. The reference work function of HOPG in air is ΦHOPG = 4.65 eV. The relative CP difference within the single scan gives the most reliable information, since on the nanometric scale the measurement is prone to unavoidable environmental pollution (adsorbate layers), tip contamination and modification, and also Φtip variability among different probes even within the same session. Accurate SKPM sessions with reproducible data allowed measurement of the work function of SLG unmodified in air (Φsample = Φtip − eVCPD), finding good agreement with that reported in literature27, ΦSLG ≈ 4.60 eV, allowing us to conclude that the CVD SLG has a good uniformity also at the nanoscale, since SKPM resolution is, in our case, approximately 60 nm. Such a high lateral resolution allows us to estimate sample uniformity and presence of residuals before and after laser machining. Fig. 6d plots averaged CPD variation along X for three different irradiation powers: 0.09, 0.12, 0.22 J/cm2. The data collected show that the damage produced by the laser pulse is well-imaged with SKPM. The LDT is identified at 0.12 ± 0.06 J/cm2, as shown in Fig. 6b–d, the ΔCP between pristine SLG and modified area becoming relevant above that value. In the area modified by pulses ranging from 0.12 to 0.22 J/cm2 the work function drops by about 100 mV. Local ablation of the SLG is achieved, as previously confirmed by Raman but, by SKPM, we identified graphitic residuals barely recognizable in topography. Along the edge (black arrows in fig. 6c) we can identify a transition region of ~0.5 μm where local increases in Φ can be explained by local folding into multi layers or a more massive accumulation of graphitic material.


Simple and effective graphene laser processing for neuron patterning application.

Lorenzoni M, Brandi F, Dante S, Giugni A, Torre B - Sci Rep (2013)

Scanning Kelvin probe force microscopy.Topography (a) and potential map of type 2 sample irradiated at (a, b) 0.12 J/cm2 and (c) 0.22 J/cm2; in (b) along a random horizontal line the ΔCP between pristine SLG and modified area is −98 ± 4 mV, while an increase of CPD is clearly visible all along the edges of the laser irradiation; between the blue arrows the increase is 50 ± 4 mV, compatible with a local folding of graphene layers; topography (a) cannot show any contrast. All images refer to a 20 × 40 μm2 area. In (d), plot of averaged CPD variation along X for three different irradiation powers: 0.09, 0.12, 0.22 J/cm2. Potential scale baselines (CPD SLG) have been set to zero. In (e) we plot two CPD profiles of the boundaries zones of scan (c), line 1 in black color is the left edge profile and line 2 in red color is the right edge profile as depicted in image (c).
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f6: Scanning Kelvin probe force microscopy.Topography (a) and potential map of type 2 sample irradiated at (a, b) 0.12 J/cm2 and (c) 0.22 J/cm2; in (b) along a random horizontal line the ΔCP between pristine SLG and modified area is −98 ± 4 mV, while an increase of CPD is clearly visible all along the edges of the laser irradiation; between the blue arrows the increase is 50 ± 4 mV, compatible with a local folding of graphene layers; topography (a) cannot show any contrast. All images refer to a 20 × 40 μm2 area. In (d), plot of averaged CPD variation along X for three different irradiation powers: 0.09, 0.12, 0.22 J/cm2. Potential scale baselines (CPD SLG) have been set to zero. In (e) we plot two CPD profiles of the boundaries zones of scan (c), line 1 in black color is the left edge profile and line 2 in red color is the right edge profile as depicted in image (c).
Mentions: Scanning Kelvin probe force microscopy measures local contact potential difference (CPD) between a conductive AFM tip and a sample. This potential difference is sensitive to local compositional and structural variations. The work function of graphene is similar to that of graphite (~4.6 eV)27 and shows a dependence on the number of layers, ranging from 4.51 eV for SLG to 4.58 eV for AB stacked HOPG28. Bi-layer and multilayer graphene should show an increase in work function of 50–100 mV due to its improved chemical stability27. Based on these considerations we performed AFM topography (Fig. 6a) and CPD scan (Fig. 6b–c) over irradiated areas. The work function of one reference tip (Φtip = 4.93 ± 0.05 eV) was calibrated on freshly cleaved highly oriented pyrolytic graphite (HOPG), as previously reported in literature29. The reference work function of HOPG in air is ΦHOPG = 4.65 eV. The relative CP difference within the single scan gives the most reliable information, since on the nanometric scale the measurement is prone to unavoidable environmental pollution (adsorbate layers), tip contamination and modification, and also Φtip variability among different probes even within the same session. Accurate SKPM sessions with reproducible data allowed measurement of the work function of SLG unmodified in air (Φsample = Φtip − eVCPD), finding good agreement with that reported in literature27, ΦSLG ≈ 4.60 eV, allowing us to conclude that the CVD SLG has a good uniformity also at the nanoscale, since SKPM resolution is, in our case, approximately 60 nm. Such a high lateral resolution allows us to estimate sample uniformity and presence of residuals before and after laser machining. Fig. 6d plots averaged CPD variation along X for three different irradiation powers: 0.09, 0.12, 0.22 J/cm2. The data collected show that the damage produced by the laser pulse is well-imaged with SKPM. The LDT is identified at 0.12 ± 0.06 J/cm2, as shown in Fig. 6b–d, the ΔCP between pristine SLG and modified area becoming relevant above that value. In the area modified by pulses ranging from 0.12 to 0.22 J/cm2 the work function drops by about 100 mV. Local ablation of the SLG is achieved, as previously confirmed by Raman but, by SKPM, we identified graphitic residuals barely recognizable in topography. Along the edge (black arrows in fig. 6c) we can identify a transition region of ~0.5 μm where local increases in Φ can be explained by local folding into multi layers or a more massive accumulation of graphitic material.

Bottom Line: Primary embryonic hippocampal neurons were cultured on our substrate, demonstrating an ordered interconnected neuron pattern mimicking the pattern design.Surprisingly, the functionalization is more effective on the SLG, resulting in notably higher alignment for neuron adhesion and growth.Therefore the proposed technique should be considered a valuable candidate to realize a new generation of highly specialized biosensors.

View Article: PubMed Central - PubMed

Affiliation: Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy. matteo.lorenzoni@iit.it

ABSTRACT
A straightforward fabrication technique to obtain patterned substrates promoting ordered neuron growth is presented. Chemical vapor deposition (CVD) single layer graphene (SLG) was machined by means of single pulse UV laser ablation technique at the lowest effective laser fluence in order to minimize laser damage effects. Patterned substrates were then coated with poly-D-lysine by means of a simple immersion in solution. Primary embryonic hippocampal neurons were cultured on our substrate, demonstrating an ordered interconnected neuron pattern mimicking the pattern design. Surprisingly, the functionalization is more effective on the SLG, resulting in notably higher alignment for neuron adhesion and growth. Therefore the proposed technique should be considered a valuable candidate to realize a new generation of highly specialized biosensors.

Show MeSH
Related in: MedlinePlus