Machine Learning-assisted Raman Spectral Analysis of Serotonin-responsive ssDNA-SWCNT Nanosensor for Improved Selectivity against Dopamine

Summary

The protocol describes Raman spectral analysis of ssDNA-wrapped SWCNT nanosensor, enabling the improved selective measurement of serotonin against dopamine with the assistance of a machine learning model.

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) plays critical roles in neuromodulation, yet current detection methods struggle to provide real-time sensing of 5-HT with high sensitivity and selectivity. We previously developed a near-infrared serotonin nanosensor (nIRHT), which consists of ssDNA-wrapped single-walled carbon nanotube that sensitively detects 5-HT. However, nIRHT’s fluorescence response cannot discriminate between 5HT and dopamine (DA), limiting its practical applications. In this study, Raman spectroscopy combined with machine learning overcomes this selectivity challenge. G-band spectral features revealed distinct signatures for 5HT versus DA binding to nIRHT, with DA causing greater G-band suppression. We employed differential Raman (ΔRaman) to isolate analyte-specific spectral changes and trained three machine learning models for classification. The random forest model with ΔRaman achieved optimal performance with 95.8% accuracy, significantly outperforming models using raw Raman spectra. This approach showed improved specificity, with negligible responses to acetylcholine, GABA, and glutamate, and achieved a detection limit of 0.1 µM suitable for physiological applications. This Raman-based approach transforms the non-selective nIRHT fluorescence sensor into a platform capable of robust neurotransmitter discrimination, overcoming selectivity issues in single-walled carbon nanotube (SWCNT)-based molecular sensing.

Introduction

Serotonin (5-hydroxytryptamine, 5-HT) is a critical neurotransmitter regulating mood, cognition, sleep, and appetite, with dysfunction implicated in depression, anxiety, and other neuropsychiatric disorders¹. Real-time monitoring of serotonin dynamics in biological systems remains challenging due to the millisecond timescale of synaptic transmission and the complex chemical environment of neural tissue². Despite significant advances in neurotransmitter detection technologies, achieving both high sensitivity and molecular selectivity for serotonin continues to pose substantial analytical challenges.

Protocol

1. ssDNA-functionalized SWCNT nIRHT nanosensor fabrication

1. Combine 1 mg of single-walled carbon nanotubes (SWCNTs), 100 µL of 1 mM E6#9 ssDNA, and 900 µL of 1x phosphate-buffered saline (PBS) in a 1.5 mL tube.
   CAUTION: Dry SWCNT powder is a potential inhalation hazard. Handle the powder inside a certified chemical fume hood and wear appropriate personal protective equipment, including a mask and gloves.
2. Disperse the mixture using a bath sonicator for 5 min. Sonicate the mixture using a tip sonicator equipped with a 3 mm probe at 50% amplitude for 30 min in an ice bath.
3. Centrifuge the sonicated solution ....

Results

The E6#9 ssDNA-functionalized SWCNT nanosensor (nIRHT), previously developed through SELEC methodology, demonstrates reversible nIR fluorescence enhancement upon 5HT binding, enabling real-time 5HT imaging in vitro and ex vivo¹². The E6#9 sequence (5'-CCCCCCAGCACCAGACAGCACACTCCCCCC-3') wraps around SWCNTs to create binding sites for 5HT. However, this sensor lacks specificity: both 5-HT and DA result in comparable intensity increase (Figure 1B), limi.......

Discussion

Raman spectroscopic analysis provided a significant enhancement in the specificity of the nIRHT sensor, a capability not achievable with its conventional fluorescence response. We observed that DA induced a more pronounced suppression of the G⁻ band (1570 cm^-1) compared to 5HT. These differential signatures likely arise from varying molecular orientations and binding geometries on the SWCNT surface. Specifically, DA's catechol moiety likely forms stronger π-π interactions with the SWCNT sur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HiPCo raw	Nanointegris
iDus1.7 InGaAs	ANDOR	DU490A-1,9
Ramantouch	Nano Photon
Vibra Cell 130	SONICS