The development of CRISPR/Cas13a-based fluorescence resonance energy transfer (FRET) beacons represents a paradigm shift in real-time microRNA detection within differentiating stem cells (Kutsche et al., 2018; Papadimitriou et al., 2023). This groundbreaking proof-of-principle study employed cytoplasmically expressed Cas13a coupled with exogenous guide RNA, though the initial signal-to-background ratio remained modest and required further optimization for clinical applications (Wang Y. et al., 2020). Recently, two significant advancements were made that substantially improved the sensitivity and temporal resolution of CRISPR-based biosensors (Wang Y. et al., 2020; Montagud-Martínez et al., 2023). First, the guide RNA (gRNA) was strategically pre-assembled with a quencher-labeled single-stranded DNA (ssDNA) reporter, creating a sophisticated detection mechanism where target binding unleashed Cas13a′s collateral cleavage activity, thereby releasing the fluorophore and significantly magnifying the detection signal (Montagud-Martínez et al., 2023; Iwasaki and Batey, 2020). Second, microfluidic integration technology confined reagents to picolitre volumes, compressing reaction time to under 15 min while maintaining high sensitivity and specificity (Degliangeli et al., 2014; Xiao et al., 2018).

The integration of CRISPR/Cas13a systems with stem cell monitoring has revolutionized understanding of microRNA dynamics during differentiation processes (Ishikawa et al., 2020). These systems enable unprecedented single-cell resolution tracking of lineage commitment, capturing the rapid molecular transitions that occur at sub-hour timescales during differentiation initiation (Lu and Yoo, 2018). The ability to detect miRNA-124, a critical neuronal differentiation marker, with such high temporal resolution has opened new avenues for studying neural stem cell fate decisions and optimizing differentiation protocols (Papadimitriou et al., 2023; Ishikawa et al., 2020).

While CRISPR beacons excel at detecting relative microRNA ratios, solid-state nanopores address the critical need for measuring absolute transcription dynamics with single-molecule precision (Li et al., 2021; Ren et al., 2025). Transition-metal dichalcogenides such as molybdenum disulfide (MoS₂) uniquely combine atomic thickness, exceptional mechanical robustness, and highly tunable electronic properties that make them ideal for nanopore applications (Xiao et al., 2018; Li et al., 2021). The sophisticated ability to temporal-stamp each individual blockade event enabled these researchers to reconstruct detailed bursty transcription kinetics in differentiating embryoid bodies with unprecedented resolution (Wheat et al., 2020). Solid-state nanopores, especially MoS₂ nanopores, allow single-molecule discrimination of transcript isoforms and real-time measurement of transcriptional bursting. Although large arrays remain in development, proof-of-concept studies demonstrate detection of individual mRNA molecules from live cell extracts, offering direct observation of stochastic gene expression that underlies iPSC fate decisions (Zhong et al., 2023). This capability reveals burst frequency and amplitude for key pluripotency genes (e.g., OCT4, SOX2) at the single-cell level.

A complementary non-destructive approach utilizes dCas9-SunTag scaffolds strategically fused to promoters of interest, providing an alternative method for monitoring transcriptional activity (Kumar et al., 2023; Papikian et al., 2019; Pflueger et al., 2018). The fluorescence signal is dramatically amplified through a sophisticated tandem array of antibody-binding peptides, with each peptide recruiting ten dye-conjugated single-chain variable fragment (scFv) components (Pflueger et al., 2018). Another research reported dCas9-CRISPR system, functioned as programmable transcriptional biosensors in ESCs, enabling real-time functional validation of microRNA promoter activity by targeting genomic regions with sgRNAs and detecting resultant changes in miRNA expression. In mouse ESCs, these tools confirmed that miR-335 expression is exclusively regulated by the host gene Mest’s promoter, demonstrating their utility for mapping lineage-specific regulatory elements in pluripotent systems (Kumar et al., 2023).

Mesenchymal stem cell osteogenic differentiation represents a cornerstone process in tissue engineering and regenerative medicine applications. Double-stranded locked nucleic acid (LNA)/DNA nanosensors enable real-time monitoring of Notch1-Dll4 signaling pathway roles at the single mesenchymal stem cell level (Zhao et al., 2022). These sensors track Dll4 mRNA expression dynamics during osteogenic differentiation, allowing observation of differentiation process heterogeneity (Zhao et al., 2022). Barium titanate nanoparticles (BT NPs) combined with alginate polymers provide a novel biocompatible three-dimensional scaffold approach for inducing osteogenic stem cell differentiation (Amaral et al., 2019). BT NP/alginate 3D scaffolds demonstrate osteogenic differentiation induction potential without requiring osteogenic supplements, with BMP-2 and ALP mRNA significantly upregulated after 21 days. The scaffolds exhibit highly interconnected pores and surface nano topography favorable for mesenchymal stem cell differentiation, with mineralization nodules formation and morphological changes from spindle to cuboid shape. Another study reported a dual-color nano sensor in which PLGA nanoparticles co-encapsulate molecular beacons for GAPDH (housekeeping control) and ALP (early osteogenic marker), enabling live, non-destructive read-out of MSC differentiation (Wiraja et al., 2016). Because the particles slowly degrade intracellularly, ALP mRNA dynamics could be followed continuously for 18 days, and the ratio metric fluorescence closely matched conventional RT-qPCR data, validating both accuracy and viability preservation. Applied in standard 2-D culture and on tricalcium-phosphate-loaded PCL films, the sensor revealed an earlier ALP peak on the osteo-inductive scaffold, demonstrating its utility for rapid screening of next-generation bone-graft materials.

Magnetic nanoparticle-enhanced extracellular vesicles (GMNPE-EVs) derived from bone marrow mesenchymal stem cells provide an effective therapeutic strategy for diabetic osteoporosis through miR-15b-5p delivery. These systems transfer miR-15b-5p to osteoclasts, downregulating GFAP expression and inhibiting osteoclast differentiation (Xu et al., 2024). In vitro experiments demonstrate that GMNPE-EVs effectively deliver miR-15b-5p to osteoclasts, while in vivo tests confirm therapeutic potential in alleviating diabetic osteoporosis. Gold nanoparticles have proven effective for mesenchymal stem cell tracking in tumor-targeted delivery systems (Xu et al., 2023). Mesenchymal stem cell-mediated gold nanoparticle delivery showed 2.4- to 9.3-fold enhanced performance in tumor accumulation and penetration compared to conventional enhanced permeability and retention (EPR) effect-based delivery.

Superparamagnetic iron oxide nanoparticles (SPIOs) serve as powerful tools for stem cell tracking applications. Cubic iron oxide nanoparticles with 22 nm edge length (CIONs-22) are optimized for magnetic particle imaging (MPI), showing superior MPI performance compared to commercialized tracers like Vivotrax (Wang Q. et al., 2020). These magnetic properties ensure high sensitivity and resolution for MPI application-ns, with efficient cellular uptake enabling real-time and prolonged monitoring of stem cells transplanted into hindlimb ischemia mice.

Electrophysiological and metabolic read-out of brain organoids has advanced far beyond occasional calcium-imaging snapshots (Yousuf et al., 2025; Park et al., 2024). Zips et al. used aerosol-jet and ink-jet additive printing to fabricate high-aspect-ratio microneedle electrode arrays composed of the conductive polymer poly (3,4-ethylenedioxythiophene):polystyrene sulfonate blended with multi-walled carbon nanotubes, creating nano-electrodes able to penetrate 400–600 µm brain spheroids and cerebral organoids (Zips et al., 2023). The microneedles had 20 µm tips, centimeter scale mechanical flexibility, and week-long electrochemical stability transform them into a practical biosensing platform that captures deep extracellular signals unreachable by conventional planar microelectrode arrays. By delivering reliable recordings from both engineered neutrosphere cultures and self-assembled organoids, the work closes a critical depth-sensing gap and illustrates how additive-manufactured nano-electrodes can advance three-dimensional neural-tissue electrophysiology. Multimodal “Phase-Zero” platforms have been reported to go a step further by threading a broadband vis/NIR fiber through the same chamber, turning the setup into a combined spectrophotometer and electrophysiology rig (Dutta et al., 2020). Local-field-potential spectral exponents in the 30–50 Hz band are logged in parallel with the optical redox read-out of cytochrome-c oxidase; together, these metrics chart excitation–inhibition balance and mitochondrial health during neurodevelopment or drug exposure in a way that single-modality systems cannot.

Mechanical, biochemical, and electrical cues in cardiac organoids require similarly integrated monitoring (Son et al., 2025). Stretchable PDMS wave-guide membranes patterned with micro-optical structures record sub-percent changes in organoid length as shifts in transmitted light intensity, enabling real-time quantification of systolic and diastolic strain under physiologically relevant after-load (Sannino et al., 2023). When human PSC-derived constructs on these membranes receive low-dose doxorubicin, contractility drops are detected hours before visible beating irregularities emerge, providing an early-warning screen for cardiotoxic compounds. On-chip electrochemical sensors add biochemical depth: aptamer-coated gold microelectrodes incorporated into the same heart-on-a-chip quantify femtomolar surges of creatine-kinase-MB and troponin-T, correlating biomarker release with beat-rate slow-down and viability loss (Devarasetty et al., 2017). At the sub-cellular scale, genetically encoded biosensors that dock a Förster-pair to the ryanodine receptor let investigators visualize cAMP transients exactly where excitation–contraction coupling begins. In β-adrenergic stimulation experiments, these reporters reveal sub-second cAMP spikes that foreshadow maladaptive remodeling, showing how optical genetics, electrochemistry, and mechanics can converge on a single micro-tissue platform.

Adding vasculature introduces new sensing demands that nanotechnology is beginning to meet (Kim J-E. et al., 2025). Electric cell–substrate impedance sensing, miniaturized onto transparent indium-tin-oxide grids, now follows tight-junction maturation in endothelial sheets derived from vascular organoids, delivering second-by-second barrier-resistance curves while leaving the epithelium available for fluorescence or trans-endothelial electric-potential imaging (Ouahoud et al., 2024; Eshetie et al., 2023; Schoon et al., 2025). Perfusable organoid-on-chip devices extend this principle: parallel microchannels lined with HUVECs are fluidically coupled to developing kidney or liver organoids, whose endogenous endothelial buds anastomose with the channel to form macro vessels (Kim Y. et al., 2025). A vascularized tumor-organoid chip grows its own micro-vessels and lets nanosensors watch endothelial sprouting, cancer cell escapes and Notch signaling live (Du et al., 2025). The open channels accept quantum-dot barcodes or plasmonic gold probes, so oxygen use, and cytokine bursts can be measured during drug tests without harming the tissue. A companion smart vascular graft places a laser-written graphene strain lattice inside flexible silicone, sensing blood-flow strain down to three ten-thousandths of a percent through more than thirty-two thousand cycles (Ma et al., 2025). The porous graphene acts both as a strain gauge and a high-area surface ready for electrochemical coatings, streaming wireless data that can flag clots or early wall thickening. Together these platforms show how nanosensors can give continuous, patient-specific feedback from both in-vitro disease models and implanted vascular devices.

Beyond brain, cardiac, and vascular organoids, nanobiosensors are increasingly being applied to other organoid types, most notably kidney, liver, and intestinal organoids, to advance disease modeling, drug screening, and tissue engineering. In kidney organoids, biosensors such as ATP/ADP ratio sensors and impedance-based devices have been integrated to enable real-time, non-invasive assessment of nephrotoxicity, transporter function, and barrier integrity, providing a sensitive platform for evaluating drug-induced injury and organoid maturation in vitro (Susa et al., 2023; Tabibzadeh and Morizane, 2024; Tabibzadeh et al., 2023). High-throughput, automated imaging systems combined with label-free biosensor algorithms further facilitate large-scale toxicity screening and functional analysis of personalized kidney organoids, achieving accuracy comparable to conventional biological assays. Organ-on-a-chip technologies have also been successfully merged with kidney and liver organoids, incorporating microfluidic flow and multisensor arrays to monitor biochemical, mechanical, and electrophysiological parameters continuously, thereby enhancing organoid maturation and physiological relevance for regenerative medicine applications (Ferrari et al., 2020; Zhang et al., 2017). Additionally, nanomaterial-based biosensors are being explored across diverse organoid models, including intestinal and tumor organoids, to monitor cell differentiation, tissue barrier function, and responses to mechanical or chemical stimuli, underscoring the broad utility of nanobiosensors in advancing 3D organoid research and translational biomedicine (Yousafzai and Hammer, 2023; Shen et al., 2023).