Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell

Abstract

Interface between neuron cells and biomaterials is the key to real-time sensing, transmitting and manipulating of neuron activities, which are the long-term pursue of scientists and gain intense research focus recently. It is of great interest to develop a sensor with exquisite sensitivity and excellent selectivity for real-time monitoring neurotransmitters transport through single live cell. Sensing techniques including electrode-based methods, optogenetics, and nanowire cell penetration systems have been developed to monitor the neuron activities. However, their biocompatibilities remain a challenge. Protein nanopores with membrane compatibility and lumen tunability provide real-time, single-molecule sensitivities for biosensing of DNA, RNA, peptides and small molecules. In this study, an engineered protein nanopore MspA (Mycobacterium smegmatis porin A) through site-directed mutation with histidine selectively bind with Cu2+ in its internal lumen. Chelation of neurotransmitters such as L-glutamate (L-Glu), dopamine (DA) and norepinephrine (NE) with the Cu2+ creates specific current signals, showing different transient current blockade and dwell time in single channel electrophysiological recording. Furthermore, the functionalized M2MspA-N91H nanopores have been embedded in live HEK293T cell membrane for real-time, in situ monitoring of extracellular L-glutamate translocating through the nanopore. This biomimetic neurotransmitter nanopore has provided a new platform for future development of neuron sensors, drug carrier and artificial synapse.

Biological nanopores for single-molecule sensing

“…Pores that exist at different stoichiometries can be triggered to assemble into different multimeric states selectively ( Fennouri et al., 2021 ) or be purified by native PAGE ( Soskine et al., 2013 ). Adapter molecules that enter the nanopore and bind inside can also be used to reduce the pores diameter and properties ( Wu et al., 2007 ; Zhang et al., 2021b ). Not only the diameter but also the length and geometry of nanopores are of interest because when reading polymers like DNA or proteins, all units (i.e., bases or amino acids) that are in or close to a nanopore constriction at a given moment contribute to the observed electrical signal, complicating its interpretation.…”

Biological nanopores for sensing applications

“…67 Combined with signal conversion and amplification, the limit of detection for Mucin 1 protein reached 0.1 fg/ml with high accuracy. Furthermore, with rational engineering and strategies, MspA was further applied to detect metal ions, 68,69 RNA structure, 24 peptide sequence, 70,71 small molecules, 72,73 and so on.…”

“…Various adapters targeted to specific molecules have been designed, including DNA aptamers, [135][136][137][138] molecular adapters, 46,61,139,140 and ion adapters. 58,72,73 For example, β-cyclodextrin (am 7 βCD) was managed to be bound covalently in α-HL mutant. With a narrower constriction, deoxynucleoside monophosphates (dNMPs) could be well distinguished.…”

A Critical Review of Carbon Quantum Dots: From Synthesis toward Applications in Electrochemical Biosensors for the Determination of a Depression-Related Neurotransmitter

“…Accordingly, for diagnosis, monitoring and therapeutic interventions for depression, it is highly desirable to detect NT concentrations in human fluids. To date there are many techniques using for detecting NTs, including nanopore, chemiluminescence, capillary electrophoresis, mass spectrometry, chromatography and electrophoresis [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. However, laborious, expensive and complicated pretreatments are the troubles that almost all methods must be able to solve.…”