In Situ Detection of Neurotransmitters from Stem Cell-Derived Neural Interface at the Single-Cell Level via Graphene-Hybrid SERS Nanobiosensing

Abstract

In situ quantitative measurements of neurotransmitter activities can provide useful insights into the underlying mechanisms of stem cell differentiation, the formation of neuronal networks, and neurodegenerative diseases. Currently, neurotransmitter detection methods suffer from poor spatial resolution, nonspecific detection, and a lack of in situ analysis. To address this challenge, herein, we first developed a graphene oxide (GO)-hybrid nanosurface-enhanced Raman scattering (SERS) array to detect dopamine (DA) in a selective and sensitive manner. Using the GO-hybrid nano-SERS array, we successfully measured a wide range of DA concentrations (10^-4 to 10^-9 M) rapidly and reliably. Moreover, the measurement of DA from differentiating neural stem cells applies to the characterization of neuronal differentiation. Given the challenges of in situ detection of neurotransmitters at the single-cell level, our developed SERS-based detection method can represent a unique tool for investigating single-cell signaling pathways associated with DA, or other neurotransmitters, and their roles in neurological processes.

Keywords: Detections of Neurotransmitters; Graphene-hybrid SERS nanobiosensing; Single-cell analysis; Surface-enhanced Raman scattering.

Figure 1.

Schematic diagram illustrating the method to detect dopamine (DA) releasing from single live cells using graphene oxide (GO)-hybrid nano-SERS. (A) Raman-enhancing graphene oxide (GO)-modified hybrid nano-surface-enhanced Raman scattering (SERS) arrays were uniformly generated on the surface of the indium tin oxide electrode via sequential laser interference lithography (LIL) and electrochemical deposition method for in situ monitoring of neural stem cell (NSC) differentiation. (B) Raman dye (malachite green)-labeled aptamer was functionalized on the surface of hybrid nano-SERS array to selectively detect DA based on SERS by electromagnetic (EM) enhancement and chemical (CM) enhancement. (C) The DA released from single dopaminergic neurons differentiated from NSC induces the detachment of Raman dye-labeled aptamers, which results in the decrease of Raman intensity on the Raman-mapping images. It enables in situ detection of DA released from single live cells.

Figure 2.

The characterization of the hybrid nano-SERS array for the enhancement of Raman intensity. (A) Schematic illustration representing the difference between the large-scale homogeneous gold nanoarrays and the conventional Raman-enhancing substrate that utilizes randomly distributed gold nanoparticles for the control experiment. (B) Helium ion microscopic (HIM) images of hybrid nano-SERS array (right) for Raman enhancement and scanning electron microscopic (SEM) image of randomly distributed spherical gold nanoparticles (left), respectively. Scale bar = 1 μm. (C) Atomic force microscopic (AFM) image of the hybrid nano-SERS array to visualize dimensions of each nanostructure generated on the surface of indium tin oxide (ITO). Scale bar = 500 nm. (D) Raman mapping images (100 μm × 80 μm, at 830 cm⁻¹) of two different GO-modified substrates with (i) hybrid nano-SERS array and (ii) randomly distributed spherical gold nanoparticles. Raman spectra were recorded using a NIR laser-emitting light at a wavelength of 785 nm, with an irradiation laser power of 3 mW for 1 s. Scale bar = 10 μm. (E) Ten different Raman spectra extracted from (D) showing D and G peaks of GO at 1400 cm⁻¹ and 1650 cm⁻¹, respectively. (F) The signal intensity and its variation obtained from the D and G peaks of GO shown in (E). (G) Top and side view of intensity (E/E₀) distributions obtained from three-dimensional finite-difference-time-domain calculations at wavelength 780 nm of hybrid structures and randomly distributed spherical gold nanoparticles. Scale bar = 500 nm.

Figure 3.

Detection of DA on the flat gold and hybrid nano-SERS array based on Raman spectroscopy. (A) Schematic diagram of the difference of the Raman intensity before and after the addition of DA on the GO modified flat gold substrate and hybrid nano-SERS array. (B, C) Raman spectra of malachite green (MG)-labeled aptamer on GO before and after addition of 1 μM DA using (B) flat gold and (C) hybrid nano-SERS array as supporting substrates. (D) Averaged intensities of four different Raman peaks of MG before and after the addition of DA. The hybrid nano-SERS array shows a clear difference in Raman intensity while the GO-modified flat gold substrate failed to show the difference between before and after the addition of DA. Raman intensities were expressed as the mean ± SEM (n = 10). Raman spectra were recorded using a NIR laser-emitting light at a wavelength of 785 nm, with an irradiation laser power of 3 mW for 1 s.

Figure 4.

Quantitative measurements of DA in the cell-free configuration and released from single dopaminergic cells. (A) MG-labeled aptamer modified hybrid nano-SERS array was utilized to detect DA based on Raman spectroscopy. Three different peaks of MG were selected as indicators to achieve the linearity of DA concentration and Raman intensity. The signal differences from negative control (0 M of DA) of (B) 830 cm⁻¹, (C) 920 cm⁻¹, and (D) 980 cm⁻¹ were obtained and normalized to GO peaks (G bands) for quantitative measurement of DA. Three different wavelengths were shown to have a high linearity value (R² ≥ 0.96) for the DA detection based on the surface-enhanced Raman resonance (SERS) signal. The standard deviation of the controls were (B) 0.026, (C) 0.016, and (D) 0.012, respectively. Data were expressed as the mean ± SEM (n = 10). (E) Schematic diagram depicting a strategy to detect DA released from single cells. Cells that released DA showed the mass detachment of Raman dye-modified aptamers on the GO surface, which contribute to a huge decrease in Raman signals that appear in Raman mapping images. (F) Representative Raman images of the (i) Raman mapping and (ii) optical image of single dopaminergic cells at 830 cm⁻¹. The optical image exactly matches with that of the Raman mapping image. Scale bars = 20 μm. (G) Raman signal intensities (830 cm⁻¹) that were obtained from different regions of Raman mapping images that appeared in (B). A single dopaminergic cell shows a clear intensity difference from each of the sections in the Raman spectra (n = 10; *p < 0.01, Student’s unpaired t-test).

Figure 5.

In situ detection of DA released from undifferentiated/differentiated NSCs. (A) Schematic diagram depicting a strategy to detect DA released from single neural stem cells, which were differentiating to the neuron for 20 days. (B) Representative immunofluorescence images of the undifferentiated/differentiated NSCs from day 0 to 20 after induction of differentiation. Scale bars = 50 μm. (C) Representative Raman mapping images corresponding to (B) at 830 cm⁻¹. Scale bars = 5 μm. The dotted lines indicate the boundary of the cells. (D) mRNA levels of the Sox-1 and MAP-2 of the differentiated NSCs, which were corresponded to (B) and (C) (n = 5; *p < 0.01, Student’s unpaired t-test). (E) Raman signal intensities (830 cm ⁻¹) that were obtained from different regions of Raman mapping images that appeared in (C) (n = 10; *p < 0.01, Student’s unpaired t-test).