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1. Introduction

Calcium ions (Ca²⁺) are an ancient and ubiquitous second messenger with diverse functions in cells in a wide array of cell types, including muscle cells, neurons, glial cells, immune cells, and oocytes. Calcium signaling plays a key regulatory role across numerous physiological processes, including cell growth, gene regulation, neuronal synaptic activity, immune system function, hormone secretion signaling, fertilization, and the biomechanics of contraction [1]. Numerous molecular pathways, such as inositol trisphosphate, adenosine triphosphate (ATP), prostaglandin E2, and nitric oxide, mediate Ca²⁺ release [2]. Tightly controlled by numerous channels and receptors, intracellular Ca²⁺ concentrations can range from nM to µM depending on the microenvironment, from approximately 100 nM in the cytoplasm to 100–800 µM in the endoplasmic reticulum (ER) to 1–2 mM in the extracellular fluid ^[3][4][5] [3–5]. Cytosolic Ca²⁺ signals can be classified as either transient (brief, small peaking due to Ca²⁺ influx from membrane channels), sustained (prolonged increase following influx from the extracellular matrix and internal Ca²⁺ stores), or oscillatory (repeated brief increases in free calcium), as defined by Uhlén and Fritz [6]. Ca²⁺ thus operates over a wide dynamic range to regulate physiological processes, triggering exocytosis of neurotransmitters over a timescale of a few hundred microseconds or driving gene transcription or cell division with sustained Ca²⁺ levels over minutes to hours ^[7][8][7,8].

Given the importance of calcium signaling in such a wide array of biological contexts, there has been an enormous amount of research devoted to unraveling the molecular players and pathways as well as the downstream effects of this activity. A crucial aspect of studying the role of intracellular Ca²⁺ in these varied processes is having robust and accurate means to detect and visualize Ca²⁺ dynamics in both in vitro and in vivo conditions. The unique spatial and temporal dynamics of free Ca²⁺ levels have necessitated high-resolution methods to facilitate detection and analysis for advancing understanding of the ubiquitous role of Ca²⁺. A successful method relies on how precisely it can bind specifically to Ca²⁺ over other ions within a proper range of concentrations, and decades have been spent focusing on developing and optimizing tools that couple fluorescent sensors with live-cell imaging.

The interconnectedness of Ca²⁺ with many molecular pathways generates complex networks within cells that establish feedback and feedforward loops, enabling multiple input signals to direct cellular level information processing, similar to modern electronic devices [9]. By using engineering principles, the goal of synthetic biology is to improve the reliability of controlling cellular behavior. User-designed inputs act together to produce a desired output in a circuit by combining standardized biological components, called “parts” [10]. These parts have been derived from natural or synthetic sources and further repurposed in recent decades to generate increasing complexity of engineered gene circuits. Synthetic biology has provided the tools needed to control and analyze endogenous biological systems, repurposing whole systems for useful functions. The majority of foundational work in synthetic biology has focused on bacteria, particularly the model organism Escherichia coli, because of its robust amenability to genetic engineering. On the eukaryotic end of the spectrum, synthetic biology has mostly focused on the yeast Saccharomyces cerevisiae, but this has brought attention to the difficulties in translating work to multicellular eukaryotes that more extensively involve RNAi, alternative splicing, and chromatin ^[11][12][13] [11–13]. However, the recent expansion of a toolkit which can incorporate RNAi-based synthetic regulators (transcription factors, RNA-binding proteins) and engineered cell-signaling components have expanded the capacity to classify mammalian cells and regulate cell fate or morphology (see reviews [14] and [9]). These tools have current and potential applications with respect to targeting intracellular calcium signaling pathways with precise spatial and temporal control while reducing off-target effects.

In covering the most recent advances in Ca²⁺ detection methods that involve calcium-responsive nanoparticles, we highlight the advantages and current limitations to establish a broad overview of the extent to which current synthetic biology tools have already been utilized to enhance study of calcium signaling and the enormous potential for synthetic biology and nanoparticle technology to enhance and expand Ca²⁺ detection in animal systems even further.

2. Calcium-Responsive Nanoparticles

2. Calcium-Responsive Nanoparticles

2.1. Overview

Nanotechnology finds applications across numerous disciplines due to the stability of various nanoparticles in different conditions and physical properties, offering a flexibility that can be applied to detect Ca

in extracellular and intracellular capacities. A key feature of in vivo systems is a reliable ability to measure Ca

dynamics in large volumes using intact tissue. Particularly in the brain, variations in extracellular [Ca

] are essential for proper synaptic activity, and nanoparticles have thus helped interrogate the voltage-induced influx of Ca

into the cell which reduces extracellular Ca

from resting levels of approximately 1 mM [175]. Relevant extracellular Ca

changes can last on the scale of tens of seconds, which can cause a drop in extracellular Ca

to concentrations as low as 100 µM. Synthetic calcium dyes and GECIs do not have the capability of detecting the relatively higher concentrations in the extracellular Ca

environment due to their higher binding affinities. While the nanoparticle Ca

detection field has developed in a niche heavily focused on extracellular Ca

, this section will cover some of these technologies which have potentially useful applications for intracellular Ca

detection as well.

2.2. Enhancing Calcium Dye Functionality and Tunability

Though molecular probes often improve aspects of performance one at a time, whether K

, ratiometric capacity, or other functionalities mentioned previously, the nanoparticle matrix has the advantage of being able to incorporate multiple agents for simultaneously tuning. The ion-selective optode design incorporates an ionophore which is a highly selective but optically silent ion carrier, a chromoionophore to serve as a fluorescent reporter, and an ion exchanger to remain electroneutrality [67]. Changing the ratio between the three components modifies the sensing range of the probe. A similar design was also applied by another set of researchers constructing fluorescent calcium-sensitive nanospheres surrounded with lipophilic matrix material to act as a chromoionophore [176]. This enabled intracellular Ca

imaging in the visible region or NIR region, such that a chromoionophore controlled the fluorescence of the nanosphere. The spectral range was adjustable depending on which chromoionophore was being chosen, and the nanosphere exhibited a rapid response time to Ca

and 24 h fluorescence.

Calcium-responsive nanoparticles can potentially enhance imaging with preexisting dyes (refer to Table 1). An important development centered on Photonic Explorers for Bioanalysis with Biologically Localized Embedding (PEBBLEs), which incorporate a fluorescent indicator such as Rhod-2 inside a nanoparticle matrix [31]. This can achieve ratiometric measurements even with non-ratiometric probes by incorporating both the sensing indicator and reference dye. The inert polymer matrix can protect the cellular environment from potential cytotoxicity of indicators while protecting the indicator from cell components. The encapsulation of the rhodamine-based fluorescent calcium indicators within the PEBBLE nanosensor provided a nanomolar dynamic sensing range for intracellular Ca

, which can be useful for measuring cytosolic free Ca

. A similar study utilized silicon nanowires (SiNWs) as substrates to anchor small molecules [177]. This configuration of a 1D fluorescence sensor utilized a red-emitting ruthenium-based dye as a reference molecule and green-emitting Fluo-3 as the response molecule to detect Ca

covalently immobilized on the surface of SiNWs. Therefore, leakage and drift of small molecule or nanoparticle-based fluorescent probes from the cell was minimized, and transfection steps that would be used for GECIs were unnecessary. A micromanipulator successfully located the SiNWs in target regions at the subcellular level, enabling recognition of differences between [Ca

] in the cell body and neurites of neurons.

Properties of calcium-responsive nanoparticles.

NR—not reported.

2.3. Magnetic Resonance Imaging of Nanoparticles

Potential avenues of exploration into imaging probes for measuring extracellular Ca²⁺ in vivo pushed researchers to find a molecularly specific analogue to functional magnetic resonance imaging (fMRI) in order to map brain activity. While fluorescent Ca²⁺ sensors have been used to measure extracellular Ca²⁺ levels, these preexisting compounds have binding constants which are too high for the targeted range of 0.1–1.0 mM and are ultimately unsuited for deep-tissue study ^[25][26][27][183–185]. To address this, Okada et al. engineered an improved MaCaReNa probe with synaptotagmin proteins, an endogenous component of synaptic neurotransmitter-release machinery which naturally respond to the narrow extracellular [Ca²⁺] fluctuations ^[15][175]. Combining the synaptotagmin 1 domains with lipid-coated iron oxide nanoparticles, MaCaReNas could be detected with MRI and display a Ca²⁺-dependent increase in the strength of a contrast agent. This shows promise as a suitable molecular-imaging paradigm for monitoring Ca²⁺ dynamics in the interstitial space of the brain over a time interval of seconds to hours. Barandov et al. also wanted to capitalize on the benefits of MRI, specifically because it offers extensive penetration depth and field of view ^[20][178]. They synthesized a manganese-based paramagnetic contrast agent, ManICS1-AM, which is designed to permeate cells and undergo esterase cleavage similar to other synthetic calcium dyes. Responses were shown to directly parallel signals obtained with fluorescent calcium indicator Fura-2FF-AM. Due to the modularity of this sensor, the membrane-permeable components, the manganese moiety, Ca²⁺-specific chelator, and the linker between them can be modified in order to optimize Ca²⁺ affinity and the paramagnetic complex interaction. This recent study can be considered the first example in which Ca²⁺ fluctuations in an in vivo rat brain were monitored directly at the intracellular level via fMRI.

To improve imaging capabilities, one study built a Ca

²⁺-binding nanoprobe which emitted a NIR fluorescence signal ^[28]. Specifically designed to have high affinity for bone minerals containing calcium phosphates, the self-assembled nanostructures could be utilized to visualize intracellular Ca

-binding nanoprobe which emitted a NIR fluorescence signal [186]. Specifically designed to have high affinity for bone minerals containing calcium phosphates, the self-assembled nanostructures could be utilized to visualize intracellular Ca

²⁺

levels in vitro and bone tissues in vivo. Macrophages and osteogenic-macrophages were treated with the nanoprobe, and NIR fluorescence signals coincided with the green fluorescence signals of the calcium indicator Fluo-3 in co-stained cells, suggesting that this nanoprobe has the potential to detect and visualize intracellular Ca

²⁺

levels. Going forward with this method will require more validation and comparison of its efficacy to other calcium indicators used to study bone tissues, especially in vivo. Another contrast agent which is bioresponsive to intracellular Ca

²⁺ at concentrations between 1 and 10 μM has recently been developed by Adams et al. ^[21]. The particular use of the conjugated NIR dye leads to a significant increase in uptake of small molecules, coincidentally improving cellular entry of the gadolinium ion (Gd

at concentrations between 1 and 10 μM has recently been developed by Adams et al. [179]. The particular use of the conjugated NIR dye leads to a significant increase in uptake of small molecules, coincidentally improving cellular entry of the gadolinium ion (Gd

³⁺

) complexes used as a contrast agent. In addition to this serendipitous event, the use of an NIR probe also allows in vitro and in vivo optical co-registration of the uptake and subcellular localization of the agent using NIR fluorescence imaging. Taken together, this multimodal MR contrast agent brings the field closer to successful study of intracellular Ca

²⁺

flux in vivo that take advantage of the benefits of MRI.

2.4. Alternative Optical Techniques to Detect Nanoparticles

2.4. Alternative Optical Techniques to Detect Nanoparticles

Near-field fluorescence (NFF) can improve the fluorescence properties of fluorophores when utilizing metal nanoparticles [4]. When the fluorophore is localized within near-field range of the metal nanoparticle surface, the excitation or emission of the fluorophore can couple with the local electromagnetic field generated by the metal nanoparticles, otherwise called the surface plasmons. In turn, excitation and emission rates are increased significantly while also extending photobleaching time, which is a significant drawback to NIR fluorophores [187]. The shaped metal nanoparticles including metal nanoshells or nanorods, such as gold nanorods (AuNR), can display their surface plasmons at longer wavelengths in the NIR region compared to spherical ones which display plasmons at the visible range [4]. When the imaging contrast agent, composed of an ICG-HSA-Au complex, was injected underneath the skin surface of mice, the luminescent nanoparticles showed 5-fold brighter emission spots compared to conjugates without the AuNRs. Consequently, AuNRs have the potential for detecting voltage-sensitive [Ca²⁺] in cells and living animals with high sensitivity.

Near-field fluorescence (NFF) can improve the fluorescence properties of fluorophores when utilizing metal nanoparticles ^[4]. When the fluorophore is localized within near-field range of the metal nanoparticle surface, the excitation or emission of the fluorophore can couple with the local electromagnetic field generated by the metal nanoparticles, otherwise called the surface plasmons. In turn, excitation and emission rates are increased significantly while also extending photobleaching time, which is a significant drawback to NIR fluorophores ^[29]. The shaped metal nanoparticles including metal nanoshells or nanorods, such as gold nanorods (AuNR), can display their surface plasmons at longer wavelengths in the NIR region compared to spherical ones which display plasmons at the visible range ^[4]. When the imaging contrast agent, composed of an ICG-HSA-Au complex, was injected underneath the skin surface of mice, the luminescent nanoparticles showed 5-fold brighter emission spots compared to conjugates without the AuNRs. Consequently, AuNRs have the potential for detecting voltage-sensitive [Ca

As seen in Section 3.3, fluorescence-resonance energy transfer (FRET) is one tool for detecting GECIs but also has more recent nanoparticle applications [181]. Building off the previously mentioned gold nanoparticle technology, researchers have recently employed a semiconductor quantum dot (QD)-gold nanoparticle which can detect Ca²⁺ inside cells upon introduction with a CPP [180]. The DSS peptide which permits successful endocytosis of the DNA aptamer-based nanoparticle has not been previously characterized but may have potential applications for introducing FRET-based Ca²⁺-detecting optical sensors intracellularly, as do other direct delivery methods that involve CPPs such as H11 [181,182]. Overall, calcium-sensitive nanoparticles are a less popular tool and have undergone less characterization for intracellular Ca

²⁺] in cells and living animals with high sensitivity.

studies. Current research nonetheless shows promising avenues for future studies on tunability and sensitivity to in vivo imaging.