Complex Biological Media Research Articles

Ultrasensitive In Vitro and Ex Vivo Tracking of 13C-Labeled PEG-PLA Degradation Products by MALDI-TOF Mass Spectrometry.

Copolymers of poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) are widely used in biomedical applications. As inactive ingredients in formulations, tracking their degradation byproducts in vivo stands as a major challenge but is a pivotal endeavor to ensure safety and further progress in clinical stages. Current bioanalytical methods used to monitor this degradation lack sensitivity and quantification precision. This study introduces a cost-effective synthetic route for 13C-labeled PEG-PLA copolymers, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to monitor their in vitro and ex vivo degradation. Incorporating 13C isotopes into copolymers significantly enhances MALDI-TOF sensitivity, allowing for precise detection of degradation products at exceedingly low concentrations. We demonstrate the ability to trace 13C-labeled PEG-PLA in complex biological media (urine, plasma) at concentrations 100 times lower than labeled PEG-PLA. Our results pave the way toward ultrasensitive in vivo tracking and elucidation of in vivo fate of this widely investigated polymer family.

Localized hybridization chain reaction amplifier utilizing three-dimensional DNA nanostructures for efficient and reliable microRNA imaging in living cells

MicroRNAs (miRNAs) are pivotal in regulating biological processes such as cell proliferation and disease progression. Traditional miRNA detection methods like qRT-PCR and Northern blotting do not allow for monitoring dynamic changes in living cells and typically require invasive sample collection. This research presents a robust, non-enzymatic technique known as the Localized Hybridization Chain Reaction Amplifier (LHCRA), designed for real-time, in vivo miRNA imaging. This method addresses these limitations by providing rapid and sensitive detection of miRNAs, crucial for progressing clinical diagnostics and research. The LHCRA utilizes hairpin chain reaction probes embedded within self-assembled DNA micelles (DM), significantly amplifying miRNA signals. LHCRA demonstrated exceptional performance, with a detection limit of 100 pM and a response time of 10 min. Importantly, it effectively differentiated between cancerous and normal cells using clinical peripheral blood samples, showcasing high specificity and stability under physiological conditions. Additionally, LHCRA maintained consistent performance in complex biological media, including 10 % serum and 2 U/mL DNase I, confirming its broad applicability in various diagnostic scenarios. This performance highlights LHCRA's potential as a transformative tool in miRNA-based diagnostics. The introduction of LHCRA marks a significant advancement in miRNA detection technology. By enabling accurate, non-invasive, and real-time monitoring of miRNA dynamics within living cells, this method offers substantial potential to enhance diagnostic accuracy and deepen our understanding of disease mechanisms, particularly in cancer. Thus, it could greatly improve therapeutic strategies and patient outcomes in clinical environment.

Fluorescence Label‐Free Doxorubicin Sensor Using Polystyrene Sulfonate as a Synthetic Receptor in Whispering Gallery Mode Microresonators

AbstractThe conventional quantification of Doxorubicin (DXR), a crucial anticancer drug, typically relies on complex and expensive techniques, limiting measurements beyond hospital settings and at different time intervals. Here, a label‐free fluorescence sensor is presented for DXR utilizing Whispering Gallery Mode (WGM) resonators. The resonators are based on fluorescent microparticles coated with a nanometer‐thick polystyrene sulfonate (PSS) layer serving as a synthetic receptor. The sensor demonstrates linear response for both position and width of the resonance peaks within the concentration range of 1–10 µg mL−1 of DXR of clinical interest. It also exhibits good selectivity against other chemotherapy drugs and can operate in complex biological fluids. The findings underscore the potential of WGM resonators as versatile multiparameter sensors for the sensitive and selective detection of small molecules, also in complex biological media.

An electrochemical biosensor with enhanced antifouling properties enabled by peptide self-assembly via robust Pt-S interactions

Designing effective biosensing interfaces requires robust biomolecule immobilization strategies. Traditional gold-sulfur bonds (Au-S) have low affinity and are prone to ligand substitution reactions in complex biological environments. In this study, we introduce an innovative approach utilizing a trifunctional branched-cyclopeptide (TBCP) for self-assembly onto platinum nanoparticles (PtNP)-modified electrodes. TBCP immobilization on PtNP via Pt-S interaction proves significantly more stable than Au-S bonding. In addition, TBCP peptides assembled through Pt-S interactions exhibit low susceptibility to displacement by small molecules like glutathione, making them ideal for constructing robust biosensors with high stability in biological fluids. Leveraging peptide multifunctionality and strong Pt-S interactions, TBCP-modified electrodes excel in various biological fluids, detecting the breast cancer marker ErbB2 in human serum. The high sensitivity of the TBCP/PtNP-based biosensor distinguishes serum samples from breast cancer patients and healthy individuals. This self-assembly strategy represents a promising advance in constructing biosensors with robust operation in complex biological media.

Leap-Type Response of Redox/Photo-Active Lanthanide-Based Metal-Organic Frameworks for Early and Accurate Screening of Prostate Cancer.

The development of high-accuracy technologies to distinguish the quite tiny concentration change of tumor markers between negative and positive is of vital significance for early screening and diagnosis of cancers, but is still a great challenge for the conventional biosensors because of their "gradual" detection mode. Herein, a unique "leap-type" responsive lanthanide MOF-based biosensor (designated as Tb-CeMOF-X) with defect-mediated redox-/photo-activities is developed for precisely identifying acid phosphatase (ACP), an early pathological marker of prostate cancer (PCa) in serum. The engineered Tb-CeMOF-X probe achieves a bursting switch-on luminescence at the critical concentration of ACP (9 U·L-1), while keeping silent below this threshold, undergoing a qualitative signal change from "zero" to "one" between negative and positive indicators and thus significantly improving the identification precision. Significantly, such "leap-type" response performance can be further edited and amplified by rational defect engineering in the crystal structure to improve the accessibility of active centers, consequently maximizing the detection sensitivity toward ACP in the complex biological media. This study proposes the first paradigm for the development of "leap-type" biosensors with ultra-sensitive differentiation capability between negative and positive,and provides a potentially valuable tool for early and accurate screening of PCa.

Rational Design of Cyanine-Based Fluorogenic Dimers to Reduce Nonspecific Interactions with Albumin and Lipid Bilayers: Application to Highly Sensitive Imaging of GPCRs in Living Cells.

Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.

Spatially confined dual-emission nanoprobes assembled from silicon nanoparticles and gold nanoclusters for ratiometric biosensing.

Gold nanoclusters (AuNCs) possess weak intrinsic fluorescence, limiting their sensitivity in biosensing applications. This study addresses these limitations by developing a spatially confined dual-emission nanoprobe composed of silicon nanoparticles (SiNPs) and AuNCs. This amplified and stabilized fluorescence mechanism overcomes the limitations associated with using AuNCs alone, achieving superior sensitivity in the sensing platform. The nanoprobe was successfully employed for ratiometric detection of bleomycin (BLM) in serum samples, operating at an excitation wavelength of 365nm, with emission wavelengths at 480nm and 580nm. The analytical performance of the system is distinguished by a linear detection range of 0-3.5μM, an impressive limit of detection (LOD) of 35.27nM, and exceptional recoveries ranging from 96.80 to 105.9%. This innovative approach significantly enhances the applicability and reliability of AuNC-based biosensing in complex biological media, highlighting its superior analytical capabilities.

S-scheme heterojunction phthalocyanine/TiO2 photoelectrochemical sensor for innovative glutathione detection.

A novel photoelectrochemical sensor, employing an S-scheme heterojunction of phthalocyanine and TiO2 nanoparticles, has been developed to enable highly sensitive determination of glutathione. By integrating the favorable stability, environmental benignity, and electronic properties of the TiO2 matrix with the unique photoactivity of phthalocyanine species, the designed sensor presents a substantial linear dynamic range and a low detection limit for the quantification of glutathione. The sensitivity is attributed to efficient charge transfer and separation across the staggered heterojunction energy levels, which generates measurable photocurrent signals. Systematic variation of phthalocyanine content reveals an optimal composition that balances light harvesting capacity and electron-hole recombination rates. The incorporation of phosphotungstic acid (PTA) in sample preparation effectively minimizes interference from compounds like L-cysteine and others. Consequently, this leads to an improvement in accuracy through the reduction of impurity levels. Appreciable photocurrent enhancements are observed upon introduction of both oxidized and reduced glutathione at the optimized composite photoanode. Coupled with advantageous features of photoelectrochemical transduction such as simplicity, cost-effectiveness, and resistance to fouling, this sensor holds great promise for practical applications in complex biological media.

Redox-responsive degradation of DNA@MnO2 nanoprobe triggering DNAzyme recycling amplification for sensitive and label-free detection of glutathione

In this work, a novel fluorescence nanoprobe based on MnO2 nanosheets and DNAzyme cyclic amplification was developed for label-free detection of GSH. In our design, the DNA@MnO2 nanoprobe was facilely prepared through the adsorption of DNA probes on the surface of MnO2 nanosheets. Importantly, MnO2 nanosheets in the DNA@MnO2 nanoprobe can act as the recognizer of analytical target GSH. In the presence of GSH, the DNA@MnO2 nanoprobe are decomposed owing to redox-responsive reaction between GSH and MnO2 nanosheets, resulting in the generation of abundant Mn2+ and the desorption of DNA probes. Subsequently, the Mn2+-dependent DNAzyme recycling amplification was rapidly initiated and a large number of the blocked G-rich sequences in DNA probes was released. The G-rich sequences were efficiently folded into G-quadruplex structures, which can remarkably light up the fluorescence intensity of thioflavin T (ThT). Experimental data demonstrated that the DNA@MnO2 nanoprobes for GSH detection have a wide linear range from 0 to 2000 µM. Moreover, this fluorescence sensing strategy was successfully applied for the detection of GSH in complex biological media with satisfying results, providing a simple, facile and cost-efficient nanoplatform for GSH-related clinical disease diagnosis.

Microbial production of 3-hydroxypropionic acid by acetic acid bacteria: Modeling including the buffering capacity of the biological medium enables prediction of pH and metabolite concentrations

A mathematical model of the fed-batch bioconversion of 1,3-propanediol into 3-hydroxypropionic acid using acetic acid bacteria is proposed. The model includes the microbial growth, the oxidation of 1,3-propanediol to 3-hydroxypropionaldehyde followed by a second oxidation reaction to 3-hydroxypropionic acid. The inhibitory effect of the total acid concentration upon the biological reactions was considered as well as the effect of pH on bacterial growth. A special attention was paid to make accurate pH predictions as pH is a key parameter that influences the microbial growth and bioconversion and also defines the strategy of downstream processing for acid recovery. The buffering capacity of the complex biological medium was found to change throughout the bioconversion. In addition to describing satisfactorily a set of experiments reported in the literature, the model was successfully used to predict metabolite concentrations and the resulting pH in new operating conditions with free pH dynamics. A sensitivity analysis was performed to identify the most influential parameters of the model. The proposed model represents a valuable tool for bioprocess design as it describes the detailed kinetics of 1,3-propanediol oxidation to 3-hydroxypropionic acid by acetic acid bacteria in bioreactor. Additionally, the pH prediction is a major feature of this model, which could guide the identification of optimal operating conditions for microbial activity with a simultaneous in-situ recovery process.

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

A low-fouling electrochemical biosensor based on BSA hydrogel doped with carbon black for the detection of cortisol in human serum

Electrochemical biosensors with high sensitivity can detect low concentrations of biomarkers, but their practical detection applications in complex biological environments such as human serum and sweat are severely limited by the biofouling. Herein, a conductive hydrogel based on bovine serum albumin (BSA) and conductive carbon black (CCB) was prepared for the construction of an antifouling biosensor. The BSA hydrogel (BSAG) was doped with CCB, and the prepared composite hydrogel exhibited good conductivity originated from the CCB and antifouling capability owing to the BSA hydrogel. An antifouling biosensor for the sensitive detection of cortisol was fabricated by drop-coating the conductive hydrogel onto a poly(3,4-ethylenedioxythiophene) (PEDOT) modified electrode and further immobilizing the cortisol aptamer. The constructed biosensor showed a linear range of 100 pg mL−1 - 10 μg mL−1 and a limit of detection of 26.0 pg mL−1 for the detection of cortisol, and it was capable of assaying cortisol accurately in complex human serum. This strategy of preparing antifouling and conductive hydrogels provides an effective way to develop robust electrochemical biosensors for biomarker detection in complex biological media.

Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Photosensitive Nanoprobes for Rapid Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations.

The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, we have developed an innovative method that utilizes photosensitive lipid nanoprobes specifically designed for efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. We demonstrate that our method provides superior capability in isolating extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 hour, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. Our method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery.

Non-enzymatic glucose sensor using mesoporous carbon screen-printed electrodes modified with cobalt phthalocyanine by phase inversion

The development of non-enzymatic glucose electrochemical sensors is still required to be used for the determination of glucose in complex biological media. This study presents a straightforward and remarkably efficient tool for the preparation of highly stable and sensitive glucose electrochemical sensors based on the deposition of cobalt phthalocyanine (CoPc) onto mesoporous carbon screen-printed electrodes (MCs). Results show that the MC electrochemical activation (aMC) followed by phase inversion (PI), which consisted of drop casting of CoPc in dimethylformamide onto a wetting electrolyte leading to the electrode aMC-CoPc/PI, enhanced sensitivity towards glucose determination in complex media. The beneficial need for MC surface activation and PI has been explored by scanning electron microscopy, energy dispersive X-ray spectroscopy, Raman spectroscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The aMC-CoPc/PI electrode exhibited the highest electrocatalytic activity of the series (namely, MC-CoPc, MC-CoPc/PI and aMC-CoPc) towards glucose oxidation. By using square wave voltammetry technique, the aMC-CoPc/PI glucose electrochemical sensor demonstrated a sensitivity of 22.3 µA mM−1 and a low detection limit of 27.4 µM (S/N = 3) in a linear dynamic range of 0.1 to 3.5 mM. Additionally, it also displayed high selectivity, robust stability, repeatability and reproducibility toward the quantification of glucose concentration in complex samples such as horse serum, intravenous glucose saline solution and culture medium for sperm cells.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.

New polymer brush-coated monodisperse magnetic nanoparticles prepared via interface-mediated RAFT polymerization for high-throughput DNA extraction from pathogen bacteria

Polymerase chain reaction (PCR) is gold-standard method for molecular detection of pathogens. DNA extraction from a complex biological medium is a crucial process for PCR based detection. Recently, magnetic nanoparticles-based methods are of great interest for genetic material isolation. In our work, we offer new magnetic nanoparticles based high-throughput DNA extraction method for pathogen detection in biofluids. For this purpose, first time polymer brush coated monodisperse magnetic nanoparticles were prepared with interface-mediated RAFT polymerization to be used in DNA extraction process. After synthesizing Fe3O4 nanoparticles, silica shell was coated on the magnetic nanoparticle core. Polymer brushes was immobilized on the amine functionalized silica layer using RAFT agent. Subsequently, the resulting particles with poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) grafted were finalized through interface-mediated RAFT polymerization. The functional groups on the nanoparticles were characterized using FTIR spectroscopy. Size distribution and zeta potential analysis of the particles were performed by TEM and Zeta-sizer respectively. Adsorption and recovery studies of plasmid DNA were carried out on the polymer-coated magnetic nanoparticles. The developed particle provided both the rapid extraction of DNA and high-yield DNA recovery without requiring extended incubation and elution time. The maximum adsorption capacity was determined as 238.1 μg/mg. About 90 % of the input DNA was recovered. The total time of DNA extraction took just 20 min. We have successfully demonstrated the extraction of dsDNA of Enterococcus faecalis spiked to reference blood sample using the developed magnetic particles. The extracted plasmid DNA and dsDNA were confirmed by gel electrophoresis and qPCR methods respectively. The results showed that the proposed magnetic iron oxide nanoparticles (MIONp) will be a strong candidate for DNA extraction process from biological matrices.

Biocompatible Ferrofluid-Based Millirobot for Tumor Photothermal Therapy in Near-Infrared-II Window.

Ferrofluidic robots with excellent deformability and controllability have been intensively studied recently. However, most of these studies are in vitro and the use of ferrofluids for in vivo medicinal applications remains a big challenge. The application of ferrofluidic robots to the body requires the solution of many key problems. In this study, biocompatibility, controllability, and tumor-killing efficacy are considered when creating a ferrofluid-based millirobot for in vivo tumor-targeted therapy. For biocompatibility problems, corn oil is used specifically for the ferrofluid robot. In addition, a control system is built that enables a 3D magnetic drive to be implemented in complex biological media. Using the photothermal conversion property of 1064nm, the ferrofluid robot can kill tumor cells in vitro; inhibit tumor volume, destroy the tumor interstitium, increase tumor cell apoptosis, and inhibit tumor cell proliferation in vivo. This study provides a reference for ferrofluid-based millirobots to achieve targeted therapies in vivo.

A DNA micro-complex containing polyaptamer for exosome separation and wound healing

Exosomes (EXOs) have showed great potential in regenerative medicine. The separation of EXOs from complex biological media is essential for the down-stream applications. Herein, we report a deoxyribonucleic acid (DNA)-based micro-complex (DMC) containing polyaptamers, which realized the specific separation of EXOs from cell culture media and the significant promotion of wound healing. The synthesis of DMCs was based on a biomineralization process via rolling circle amplification (RCA) under the catalysis of phi29 DNA polymerase. To endow DMCs with the ability to capture EXOs, the DNA template of RCA was integrated with complementary sequence of aptamer that specifically recognized the CD63 proteins on EXOs. The obtained DMCs contained polyaptamers that can specifically capture the EXOs in cell culture media. The EXOs-capturing DMCs were collected by centrifugation, achieving the separation of EXOs. Mesenchymal stem cell (MSC)-derived EXOs (MSC-EXOs) were separated by this DMC-based strategy, and the separated MSC-EXOs significantly enhanced the migration ability of cells. In particular, the significant therapeutic efficacy of the DMCs with MSC-EXOs was verified in full-thickness wound excision mouse models, in which the wounds completely healed in 10 days. We envision that this DMC-based separation strategy can be a promising route to promote the development of EXOs in biomedicine.

Medical Imaging Technology for Micro/Nanorobots.

Due to their enormous potential to be navigated through complex biological media or narrow capillaries, microrobots have demonstrated their potential in a variety of biomedical applications, such as assisted fertilization, targeted drug delivery, tissue repair, and regeneration. Numerous initial studies have been conducted to demonstrate the biomedical applications in test tubes and in vitro environments. Microrobots can reach human areas that are difficult to reach by existing medical devices through precise navigation. Medical imaging technology is essential for locating and tracking this small treatment machine for evaluation. This article discusses the progress of imaging in tracking the imaging of micro and nano robots in vivo and analyzes the current status of imaging technology for microrobots. The working principle and imaging parameters (temporal resolution, spatial resolution, and penetration depth) of each imaging technology are discussed in depth.