Designing Recognition Molecules and Tailoring Functional Surfaces for In Vivo Monitoring of Small Molecules in the Brain

ConspectusThe in vivo analysis of chemical signals in brain extracellular fluid (ECF) using implanted electrochemical biosensors is a vital way to study brain functions and brain activity mapping. This approach offers excellent spatial (10–200 μm) and temporal (approximately second) resolution and the major advantage of long-term stability. By implantation of a microelectrode in a specific brain region, changes in the concentration of a variety of ECF chemical species can be monitored through applying a suitable electrical signal and, typically, recording the resulting Faradaic current. However, the high performance requirements for in vivo biosensors greatly limit our understanding of the roles that biomolecules play in the brain. Since a large number of biological species, including reactive oxygen species (ROS), metal ions, amino acids, and proteins, coexist in the brain and interact with each other, developing in vivo biosensors with high selectivity is a great challenge. Meanwhile, it is difficult to quantitatively determine target molecules in the brain because of the variation in the distinct environments for monitoring biomolecules in vitro and in vivo. Thus, there are large errors in the quantification of concentrations in the brain using calibration curves obtained in artificial cerebrospinal fluid (aCSF). More importantly, to gain a full understanding of the physiological and pathological processes in the brain, the development of novel approaches for the simultaneous determination of multiple species in vivo is urgently needed.This Account provides insight into the basic design principles and criteria required to convert chemical/electrochemical reactions into electric signals, while satisfying the increasing requirements, including high selectivity, sensitivity, and accuracy, for the in vivo analysis of biomolecules in the brain. Recent developments in designing various functional surfaces, such as self-assembled monolayers, gold nanostructures, and nanostructured semiconductors for facilitating electron transfer from specific enzymes, including superoxide dismutase (SOD), and further application to an O₂^•– biosensor are summarized. This Account also aims to highlight the design principles for the selective biosensing of Cu²⁺ and pH in the brain through the rational design and synthesis of specific recognition molecules. Additionally, electrochemical ratiometric biosensors with current signal output have been constructed to correct the effect of distinct environments in a timely manner, thus greatly improving the accuracy of the determination of Cu²⁺ in the live brain. This method of using a built-in element has been extended to biosensors with the potential signal output for in vivo pH analysis. More importantly, the new concept of both current and potential signal outputs provides an avenue to simultaneously determine dual species in the brain.The extension of the design principles and developed strategy demonstrated in this Account to other biomolecules, which may be closely correlated to the biological processes of brain events, is promising. The final section of this Account outlines potential future directions in tailoring functional surfaces and designing recognition molecules based on recent advances in molecular science, nanoscience and nanotechnology, and biological chemistry for the design of advanced devices with multiple target species to map the molecular imaging of the brain. There are still opportunities to engineer surfaces that improve on this approach by constructing implantable, multifunctional nanodevices that promise to combine the benefits of multiple sensing and therapeutic modules

Micro Electrochemical pH Sensor Applicable for Real-Time Ratiometric Monitoring of pH Values in Rat Brains

To develop in vivo monitoring meter for pH measurements is still the bottleneck for understanding the role of pH plays in the brain diseases. In this work, a selective and sensitive electrochemical pH meter was developed for real-time ratiometric monitoring of pH in different regions of rat brains upon ischemia. First, 1,2-naphthoquinone (1,2-NQ) was employed and optimized as a selective pH recognition element to establish a 2H⁺/2e^– approach over a wide range of pH from 5.8 to 8.0. The pH meter demonstrated remarkable selectivity toward pH detection against metal ions, amino acids, reactive oxygen species, and other biological species in the brain. Meanwhile, an inner reference, 6-(ferrocenyl)­hexanethiol (FcHT), was selected as a built-in correction to avoid the environmental effect through coimmobilization with 1,2-NQ. In addition, three-dimensional gold nanoleaves were electrodeposited onto the electrode surface to amplify the signal by ∼4.0-fold and the measurement was achieved down to 0.07 pH. Finally, combined with the microelectrode technique, the microelectrochemical pH meter was directly implanted into brain regions including the striatum, hippocampus, and cortex and successfully applied in real-time monitoring of pH values in these regions of brain followed by global cerebral ischemia. The results demonstrated that pH values were estimated to 7.21 ± 0.05, 7.13 ± 0.09, and 7.27 ± 0.06 in the striatum, hippocampus, and cortex in the rat brains, respectively, in normal conditions. However, pH decreased to 6.75 ± 0.07 and 6.52 ± 0.03 in the striatum and hippocampus, upon global cerebral ischemia, while a negligible pH change was obtained in the cortex

Mitochondria-Targeted Ratiometric Fluorescent Nanosensor for Simultaneous Biosensing and Imaging of O₂^•– and pH in Live Cells

Intracellular pH undertakes critical functions in the formation of a proton gradient and electrochemical potential that drives the adenosine triphosphate synthesis. It is also involved in various metabolic processes occurring in mitochondria, such as the generation of reactive oxygen species, calcium regulation, as well as the triggering of cell proliferation and apoptosis. Meanwhile, the aberrant accumulation of O₂^•– within mitochondria is frequently intertwined with mitochondrial dysfunction and disease development. To disentangle the complicated inter-relationship between pH and O₂^•– in the signal transduction and homeostasis in mitochondria, herein we developed a mitochondria-targeted single fluorescent probe for simultaneous sensing and imaging of pH and O₂^•– in mitochondria. CdSe/ZnS quantum dots encapsulated in silica shell was designed as an inner reference element for providing a built-in correction, as well as employed as a carrier to assemble the responsive elements for O₂^•– and pH, together with mitochondria-targeted molecule. The developed nanosensor demonstrated high accuracy and selectivity for pH and O₂^•– sensing, against other ROS, metal ions, and amino acids. The remarkable analytical performance of the present nanosensor, as well as good biocompatibility, established an accurate and selective approach for real-time imaging and biosensing of O₂^•– and pH in mitochondria of live cells

Ratiometric Electrochemical Sensor for Selective Monitoring of Cadmium Ions Using Biomolecular Recognition

A selective, accurate, and sensitive method for monitoring of cadmium ions (Cd²⁺) based on a ratiometric electrochemical sensor was developed, by simultaneously modifying with protoporphyrin IX and 6-(ferroceney) hexanethiol (FcHT) on Au particle-deposited glassy carbon electrode. On the basis of high affinity of biomolecular recognition between protoporphyrin IX and Cd²⁺, the functionalized electrode showed high selectivity toward Cd²⁺ over other metal ions such as Cu²⁺, Fe³⁺, Ca²⁺, and so on. Electroactive FcHT played the role as the inner reference element to provide a built-in correction, thus improving the accuracy for determination of Cd²⁺ in the complicated environments. The sensitivity of the electrochemical sensor for Cd²⁺ was enhanced by ∼3-fold through the signal amplification of electrodeposited gold nanoparticles. Accordingly, the present ratiometric method demonstrated high sensitivity, broad linear range from 100 nM to 10 μM, and low detection limit down to 10 nM (2.2 ppb), lower than EPA and WHO guidelines. Finally, the ratiometric electrochemical sensor was successfully applied in the determination of Cd²⁺ in water samples, and the obtained results agreed well with those obtained by the conventional ICP-MS method

Label-Free Electrochemical Biosensor for Monitoring of Chloride Ion in an Animal Model of Alzhemier’s Disease

The potential damage of Alzheimer’s disease (AD) in brain function has attracted extensive attention. As the most common anion, Cl^– has been indicated to play significant roles in brain diseases, particularly in the pathological process of AD. In this work, a label-free selective and accurate electrochemical biosensor was first developed for real-time monitoring of Cl^– levels in a mouse brain model of AD and rat brain upon global cerebral ischemia. Silver nanoparticles (AgNPs) were designed and synthesized as selective recognition element for Cl^–, while 5′-MB-GGC­GCG­ATT­TT-SH-3′ (SH-DNA-MB, MB = methylene blue) was selected as an inner reference molecule for a built-in correction to avoid the effects from the complicated brain. The electrochemical biosensor showed high accuracy and remarkable selectivity for determination of Cl^– over other anions, metal ions, amino acids, and other biomolecules. Furthermore, three-dimensional nanostructures composed of single-walled carbon nanotubes (SWNTs) and Au nanoleaves were assembled on the carbon fiber microelectrode (CFME) surface to enhance the response signal. Finally, the developed biosensor with high analytical performance, as well as the unique characteristic of CFME itself including inertness in live brain and good biocompatibility, was successfully applied to in vivo determination of Cl^– levels in three brain regions: striatum, hippocampus, and cortex of live mouse and rat brains. The comparison of average levels of Cl^– in normal striatum, hippocampus, and cortex of normal mouse brains and those in the mouse model brains of AD was reported. In addition, the results in rat brains followed by cerebral ischemia demonstrated that the concentrations of Cl^– decreased by 19.8 ± 0.5% (<i>n</i> = 5) in the striatum and 27.2 ± 0.3% (<i>n</i> = 5) in hippocampus after cerebral ischemia for 30 min, but that negligible change in Cl^– concentration was observed in cortex

Two-Photon Ratiometric Fluorescence Probe with Enhanced Absorption Cross Section for Imaging and Biosensing of Zinc Ions in Hippocampal Tissue and Zebrafish

Zinc ion (Zn²⁺) not only plays an important function in the structural, catalytic, transcription, and regulatory of proteins, but is also an essential ionic signal to regulate brain neurotransmitters pass process. In this work, we designed and synthesized an intramolecular charge transfer-based ratiometric two-photon fluorescence probe, P–Zn, for imaging and biosensing of Zn²⁺ in live cell, hippocampal tissue, and zebrafish. The developed probe demonstrated high two-photon absorption cross section (δ) of 516 ± 77 GM, which increased to 958 ± 144 GM after the probe was coordinated with Zn²⁺. Furthermore, this P–Zn probe quickly recognized Zn²⁺ with high selectivity, over other metal ions, amino acids, and reactive oxygen species. More interestingly, the initial emission peak of the present probe at 465 nm decreased with a new peak increased at 550 nm, leading to the ratiometric determination of Zn²⁺ with high accuracy. Finally, this two-photon fluorescence probe with high temporal resolution and remarkable analytical performance, as well as low-cytotoxicity, was successfully applied in imaging of live cells, hippocampal tissues, and zebrafishes. The present P–Zn probe combined with FLIM provided accurate mapping of Zn²⁺ distribution at single-cell level. More interestingly, the two-photon spectroscopic results demonstrated that the level of Zn²⁺ in hippocampal tissue of mouse with AD was higher than that in normal mouse brain

A Single Nanoprobe for Ratiometric Imaging and Biosensing of Hypochlorite and Glutathione in Live Cells Using Surface-Enhanced Raman Scattering

Hypochlorite (ClO^–) and glutathione (GSH) have been reported to closely correlate with oxidative stress and related diseases; however, a clear mechanism is still unknown, mainly owing to a lack of accurate analytical methods for live cells. Herein we create a novel surface-enhanced Raman scattering (SERS) nanoprobe, 4-mercaptophenol (4-MP)-functionalized gold flowers (AuF/MP), for imaging and biosensing of ClO^– and GSH in RAW 264.7 macrophage cells upon oxidative stress. The SERS spectra of AuF/MP change with the reaction between ClO^– and 4-MP on AuFs within 1 min and then recover after reaction with GSH, resulting in the ratiometric detection of ClO^– and GSH with high accuracy. The single SERS probe also shows high selectivity for ClO^– and GSH detection against other reactive oxygen species and amino acids which may exist in biological systems, as well as remarkable sensitivity ascribed to a larger amount of hot spots on AuFs. The significant analytical performance of the developed nanoprobe, together with good biocompatibility and high cell-permeability, enables the present SERS probe imaging and real-time detection of ClO^– and GSH in live cells upon oxidative stress

Single Probe for Imaging and Biosensing of pH, Cu²⁺ Ions, and pH/Cu²⁺ in Live Cells with Ratiometric Fluorescence Signals

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu²⁺, to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu²⁺, and pH/Cu²⁺ with different ratiometric fluorescent signals. First, 2,2′,2″-(2,2′,2″-nitrilotris­(ethane-2,1-diyl)­tris­((pyridin-2-yl-methyl)­azanediyl))­triethanethiol (TPAASH) was designed for specific recognition of Cu²⁺, as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu²⁺, and pH/Cu²⁺ excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu²⁺ against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic–organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu²⁺ and in understanding the interplaying roles of Cu²⁺ and pH in live cells by ratiometric multicolor fluorescent imaging

Carbon-Dot-Based Ratiometric Fluorescent Probe for Imaging and Biosensing of Superoxide Anion in Live Cells

In this article, a ratiometric fluorescent biosensor for O₂^•– was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O₂^•–, playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O₂^•–, a new emission peak ascribed to the reaction products of HE and O₂^•– was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O₂^•– but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O₂^•–. The inorganic–organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ∼5 × 10^–7–1.4 × 10^–4 M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O₂^•– over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic–organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O₂^•– changes upon oxidative stress

Bioimaging and Biosensing of Ferrous Ion in Neurons and HepG2 Cells upon Oxidative Stress

Iron ions, as a main component of intracellular labile iron, not only play an important function in oxygen transport, enzymatic reactions, and electron transport but also are vitally important in oxidative stress. In this work, we developed a ratiometric fluorescent biosensor for ferrous ion (Fe²⁺), in which gold nanoclusters (AuNCs) were synthesized as a stable fluorescent probe and a ligand (FeL) was designed for specific recognition of Fe²⁺ and conjugated onto AuNCs (AuNC@FeL). Meanwhile, water-soluble sulfocyanine 7 <i>N</i>-hydroxysuccinimide ester (Cy7 NHS ester) was immobilized onto AuNC@FeL as a reference element. The developed ratiometric fluorescent nansosensor displayed good linearity with the concentration of Fe²⁺ in the range of 1–105 μM, and detection limit was achieved down to 210 nM. In addition, this nanosensor responded to Fe²⁺ in less than 1.23 s and showed high selectivity against other metal ions, amino acids, and reactive oxygen species. With the advantages of high selectivity and accuracy, as well as quick response and long-term stability, this organic–inorganic ratiometric fluorescent probe was successfully applied in real-time biosensing and bioimaging of Fe²⁺ in neurons and HepG2 cells. By use of this tool, it was found that the increasing concentration of Fe²⁺ in live cells was closely related to oxidative stress