Hypersensitive detection of single millimeter vascular emboli from adhesive in vivo

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: minor_revision

Abstract
Surgical adhesives are widely used in clinical practice but pose a significant risk of severe vascular embolism complications. Nevertheless, there are currently no non-invasive direct methods for precise detection of detached emboli.

Herein, we show a CT-visualized method for hypersensitive detection of single millimeter vascular emboli from adhesive in vivo by simply doping BiOCl into surgical adhesives. As proof of concept, BiOCl-BioGlue with excellent CT imaging capability is fabricated and applied to repair ruptured vessels and liver in male rats. The location, morphology, and degradation process of BiOCl-BioGlue can be dynamically monitored by CT imaging for 42 days, and pulmonary emboli caused by BiOCl-BioGlue, with sizes as small as 1.2 mm, are successfully detected. Additionally, the high K-edge of Bi enables precise detection of pulmonary emboli in spectral CT imaging, unaffected by confounding calcifications. The proposed non-invasive detection strategy for adhesive emboli significantly enhances the biosafety of surgical adhesives.

Introduction
Surgical adhesives, such as the FDA-approved BioGlue, TISSEEL, Progel, and PreveLeak, offer several advantages, including reducing operative time, decreasing bleeding, minimizing tissue damage, lowering infection risk, enhancing patient comfort, and diminishing scar formation. They are widely utilized in clinical practice for hemostasis, reinforcement of suture lines, and the repair of fragile tissues, significantly enhancing the safety and efficacy of surgical procedures1–3. Despite significant clinical benefits, the leakage and detachment of surgical adhesives at suture lines may lead to severe complication of vascular embolism4,5. As the most extensively used surgical adhesive, BioGlue poses a risk of generating adhesive-derived emboli during and after surgery, and embolism commonly occurs in pulmonary artery, coronary artery, and cerebral vessels in clinic6–10. Adhesive-derived emboli with different sizes (ranging from 1.0 mm to several millimeters) may affect tissue perfusion, leading to ischemia and necrosis, and can even result in cerebral infarction and myocardial infarction, potentially endangering life. Besides, adhesive-derived emboli cannot be effectively treated by conventional anticoagulant or thrombolytic therapies and typically require interventional or surgical removal. Thus, early and accurate detection of the embolus is crucial for timely treatment of vascular embolism complication.

Computed tomography angiography (CTA) and Digital subtraction angiography (DSA) are most used imaging methods for detecting vascular embolism in clinic, which diagnoses embolism by detecting obstructed or interrupted contrast agent flow11–13. As contrast agent injection is required, they are typically performed only when patients exhibit clinical symptoms, resulting in difficulties in early detection and missing the optimal treatment opportunities. Moreover, CTA and DSA are indirect methods for embolism detection, which is unable to directly visualize embolus or classify their origins. In recent years, intravascular ultrasound (IVUS) and optical coherence tomography (OCT) are employed for directly visualizing BioGlue-induced embolus successfully14,15. These two methods are invasive, have limited penetration depth, and are not commonly utilized in clinical examinations. Therefore, it is of great significance to develop new methods for the noninvasively precise detection of adhesive-induced vascular embolism.

Incorporating imaging units into adhesives or gels is a commonly employed strategy for visualizing them through various imaging techniques like computed tomography (CT) imaging16–19, magnetic resonance imaging20, nuclear imaging17,21, ultrasound imaging22–24, and photoacoustic imaging25–27. These approaches have been extensively utilized to detect the location, morphology, and degradation behavior of adhesives and gels, and has found widespread application in tissue engineering, disease diagnosis, drug delivery, and immunotherapy. Inspired by these advanced developments, incorporating imaging probes into adhesives provides a valuable opportunity for the noninvasively precise detection of adhesive-induced emboli. However, there are currently no relevant studies to our best knowledge.

In this work, we presented an ultra simple strategy to endow surgical adhesives with CT visualization capability for hypersensitive detection of single millimeter vascular emboli from adhesive in vivo for the first time (Fig. 1). The CT-visualized adhesive was synthesized by incorporating amorphous BiOCl with excellent X-ray absorption property and good compatibility with adhesive via a straightforward and universal method. The introduction of BiOCl allowed various adhesives to achieve remarkable CT imaging capability without compromising their gelation rate and adhesion strength. Using BiOCl-doped adhesive as an example, we conducted a systematic study on wound adhesion, adhesive monitoring, and detached embolus detection in vivo. The Bi-BioGlue demonstrated high efficacy in repairing ruptured blood vessel and liver, with its location and degradation dynamically monitored by CT imaging over a period of 42 days. Furthermore, the embolus as small as 1.2 mm, derived from Bi-BioGlue, was readily detected by CT imaging in a pulmonary embolism model. Additionally, due to the high K-edge energy value of Bi, the embolus was precisely distinguished from calcifications within the lungs using spectral CT imaging. Our study provided a noninvasive, precise and universal method for the early detection of adhesive-associated embolus, thereby significantly improving the biosafety of surgical adhesives.

Results
Synthesis and Characterization of CT-visualized Adhesives
Among various imaging modalities, CT imaging is routinely used in clinical settings due to its deep tissue penetration, high density resolution, and fast imaging speed, making it exceptionally suitable for the detection of emboli28,29. However, due to the small size of emboli and their uncertain distribution within the body, the CT imaging contrast agent used to label surgical adhesives must possess high sensitivity and good biocompatibility. Compared to the widely used iodine-based CT contrast agents with lower sensitivity, elements with high atomic numbers and greater X-ray absorption capabilities are increasingly utilized to develop CT contrast agents with enhanced sensitivity. Notably, as bismuth (Bi) element possess highest atomic number in all non-radioactive elements, thus exhibiting superior X-ray attenuation ability30. In addition, Bi, the most cost-effective high atomic number element for constructing CT imaging probes31–38, offers good biocompatibility and many Bi-drugs, like colloidal bismuth subcitrate, ranitidine bismuth citrate, and bismuth subsalicylate are widely used in basic research and in clinic39–45. Therefore, it is an ideal choice to introduce Bi-based probe into surgical adhesives, combined with CT imaging for early and precise detection of embolism complication.

BioGlue is composed of purified bovine serum albumin (BSA, 45%, w/v) and glutaraldehyde (GA, 10%, w/v), which are mixed in a dual-barrel syringe and applied to the surgical site. The principle behind BioGlue is that glutaraldehyde forms a stable seal by covalently linking to the lysine amino acid fragment in BSA, and it can also connect to lysine residues in other proteins in the tissue, resulting in excellent tissue adhesion (Fig. 2a). The strategy of incorporating imaging probes into surgical adhesives require that the probes are evenly distributed within the adhesive and have an appropriate labeling duration to ensure optimal CT imaging results and an extended imaging time window. We selected several Bi-based imaging probes with varying physicochemical properties, including completely water-soluble Bi-DTPA small molecules (Fig. S1a), BiOCl flocculent precipitates (Fig. S1b), fully hydrophobic Bi2S3 and Bi2O3 powders, to investigate their effects on labeling the most commonly used clinical adhesive, BioGlue. Bi-DTPA was synthesized according to our previously reported method46 (Fig. S1c), BiOCl, composed of aggregated nanoparticles with poor water solubility, was fabricated using a simple hydrolysis method (Fig. S1d, e), and Bi2O3 and Bi2S3 were obtained through commercial sources. The Bi-based BioGlue was synthesized facilely by mixing Bi-based probes with a BSA solution, followed by the addition of GA. Bi-based BioGlue and BioGlue share the same crosslinking mechanism, which is based on the covalent interaction between BSA and GA. The only difference lies in the composition of the protein component: Bi-based BioGlue incorporates BSA doped with Bi-based imaging probes, whereas BioGlue utilizes native BSA (Fig. 2a). The GA can cross-link with BSA via Schiff base formation between its aldehyde groups and amino groups in BSA. All four types of Bi-based probes were doped into BSA solution and successfully formed gels upon incorporation with GA (Fig. 2b). CT images revealed that all four Bi-based BioGlue samples exhibited significant contrast enhancement compared to the blank BioGlue. However, the contrast enhancement in the BioGlue doped with Bi2S3 and Bi2O3 was notably uneven, attributed to the strong hydrophobicity of Bi2S3 and Bi2O3 materials, causing their uneven distribution within the adhesive (Fig. 2b, c). In contrast, the uniform CT enhancement indicated that BiOCl and Bi-DTPA can be evenly dispersed in BioGlue. However, the CT value of Bi-DTPA-BioGlue decreased sharply within 5 h when soaked in water, due to the small molecular size and good water solubility of Bi-DTPA, which allowed it to rapidly diffuse from BioGlue into the surrounding solution. In the same conditions, the CT value of BiOCl-BioGlue remained stable over 24 h. These results suggested that, considering both labeling uniformity and persistence, BiOCl provided the optimal labeling effect for the adhesive (Fig. 2d).

Bi doping concentration was systematically optimized to assess its effect on the CT imaging performance of Bi-BioGlue. Increasing the Bi content enhanced the sensitivity of CT detection; however, excessive doping led to undesirable changes in the colloidal properties and raised potential concerns about biosafety. The result showed that the CT value of bulk Bi-BioGlue gradually increased with rising Bi concentrations. When the Bi concentration reached 105 mM, the CT value of Bi-BioGlue reached approximately 659 HU, yielding a favorable signal-to-background ratio compared to normal tissues, which typically exhibit CT values in the range of 40-80 HU. It should be noted that maintaining a high CT value in bulk Bi-BioGlue relative to soft tissue is essential, as the formation of very small BioGlue emboli will lead to a certain degree of CT signal attenuation due to partial volume effects. Therefore, 105 mM was selected as the optimal Bi doping concentration for the following studies.

Besides BioGlue, BiOCl was further attempted to be incorporated into other types of adhesives, including gelatin and sodium alginate adhesives47–49. All BiOCl-doped gels exhibited the same gelation process as the corresponding blank gels and demonstrated similar CT contrast enhancement effects as Bi-BioGlue, confirming the good versatility of the BiOCl doping strategy in surgical adhesives (Fig. 2e and Fig. S2).

The amide I band vibrations at 1655 cm-1 and amide II band vibrations at 1540 cm-1 in BSA were both observed in the FTIR spectrum of the BioGlue, Bi-BioGlue, and characteristic O=CH- stretching vibration at 1710 cm-1 for GA disappeared after the crosslinking reaction with BSA, indicating the successful synthesis of BioGlue and Bi-BioGlue (Fig. 2f)50. Elemental mapping images demonstrated the presence and uniform distribution of Cl (orange), Bi (yellow), and S (purple) in the freeze-dried Bi-BioGlue (Fig. 2g), proving the homogeneous distribution of BiOCl in as-prepared Bi-BioGlue. Furthermore, the synthesis strategy can be extended to the preparation of other metal (Hf/Ta/Yb)-doped BioGlue (Fig. S3). Dynamic oscillation time sweep measurements showed that exhibit similar G’/G” ratios, indicating that doping with metal elements does not affect the rheological properties of the adhesives (Fig. S4). The crosslinking kinetics of both BioGlue and Bi-BioGlue by rheological analysis demonstrated that upon mixing, the BSA and glutaraldehyde components exhibited similarly rapid crosslinking kinetics, forming an opaque white solid within seconds (Fig. 2h). The Bi-BSA mixture and GA could be easily extruded from the needle to form Bi-BioGlue with customizable pattern (e.g., TMU) with good CT imaging capacity (Fig. 2i). In addition, the morphological changes of Bi-BioGlue after incubation in various simulated physiological media over different time periods and the release of Bi ions were systematically studied. The experimental results demonstrated that Bi-BioGlue maintained a stable morphology throughout the monitoring period, with negligible Bi3+ release (Fig. S5, S6). Further XRD analysis showed that BiOCl, whether incorporated into Bi-BioGlue or not, retained its crystalline structure across all media without noticeable changes (Fig. S7). These findings indicate that BiOCl exhibits efficient labeling within the BioGlue matrix, and the resulting Bi-BioGlue possesses excellent physicochemical stability.

In Vitro Conventional/Spectral CT Imaging
For comparison, clinically used iodine-based contrast agents was also doped into BioGlue (I-BioGlue). Though CT intensity of both Bi-BioGlue and I-BioGlue was linear enhancement with the increasement of concentration, the Bi-BioGlue yielded a more pronounced CT image brightness and higher CT intensity compared to I-BioGlue on same Bi/I-doped concentration at commonly used tube voltage in clinic (120 kV) (Fig. 2e and Fig. S8). In addition, since clinical iodine-based contrast agents are small molecules with excellent water solubility, I-BioGlue exhibited a burst release behavior of imaging probes similar to that of Bi-DTPA-BioGlue when immersed in water. These results indicated that clinical iodine-based contrast agents are unsuitable for labeling surgical adhesives due to their relatively poor sensitivity and labeling persistence (Fig. S9).

Traditional CT imaging uses a single X-ray tube emitting mixed-energy X-rays, producing images based on the absorption of these mixed-energy X-rays by the scanned object. In contrast, spectral CT is an advanced imaging technology that utilizes two X-ray tubes operating at high and low tube voltages. Through computer analysis and processing, spectral CT generates images based on the absorption of single-energy X-rays by the scanned object. Spectral CT offers unique capabilities, such as monochromatic imaging, material separation, effective atomic number determination, and removal of metal artifacts51–55. Due to the much higher K-edge energy value of Bi (91 keV) compared to the I (33.2 keV), the CT intensity of I-BioGlue showed sharply attenuation decay curve and at high monochromatic X-ray energy on spectral CT imaging, while the Bi-BioGlue exhibited a relatively flat decay curve. At 190 keV, the CT value of Bi-BioGlue was 3.9 times higher than that of Iohexol-doped BioGlue. The results demonstrated that Bi-BioGlue exhibited better contrast effects compared to clinical iohexol (Fig. 2j, k).

Adhesion Ability of Bi-BioGlue
Thereafter, we investigated the adhesion performance of Bi-BioGlue in vitro. The injection apparatus consists of glue gun, bayonet mixing tube and 50 mL double cylinder, which can mix and inject the BiOCl-BSA and GA in a volume ratio of 4:1 (Fig. 3a). Due to the plenty of amine groups in BSA and biological tissues, the GA can interact with them by crosslink reaction to realize adhesion. To evaluate the adhesive performance, BioGlue and Bi-BioGlue were applied in the interlayer of two porcine aortic vessels (about 1 cm2), respectively. The vessels were tightly connected by Bi-BioGlue and BioGlue, with maximum withstandable pulling forces of 4.88 N and 4.87 N, respectively, indicating that the adhesive strength between Bi-BioGlue and BioGlue is comparable (Fig. 3b). Then, an ex vivo porcine aorta adhesion test was carried out to further evaluate the adhesive ability of Bi-BioGlue. The aorta’s lower end was tied off with sutures, and a gap was created at the aortic bifurcation. When red dye simulated blood flowed into the upper end, and fluid outflow from the gap was observed. After connecting the vessel gap using Bi-BioGlue, the fluid outflow ceased completely, thus confirming the successful closure of the gap (Fig. S10).

In Vivo Monitoring of Bi-BioGlue by CT Imaging
Next, the metabolic process of Bi-BioGlue was investigated by CT imaging. 30 μL of Bi-BioGlue was surgically adhered to the posterior vena cava of SD rats, and CT imaging revealed significant contrast enhancement at the adhesion site, with a CT value as high as 260 HU. The Bi-BioGlue maintained stable morphology and size within the first week, with only a gradual decrease in CT signal intensity. This indicates that the initial degradation of Bi-BioGlue is very slow and that there is no apparent presence of unlabeled adhesive. As time progressed, Bi-BioGlue exhibited further degradation, as evidenced by a gradual reduction in size on CT imaging, eventually the CT value of adhesion site returning to the soft tissue level (40 HU) by day 42 (Fig. 3c, d). Meanwhile, HE staining confirmed almost complete degradation of Bi-BioGlue in the adhesive region (Fig. 3c). The above results indicated that Bi-BioGlue possessed a relatively prolonged imaging time window and could undergo gradual degradation in vivo. Given the negligible Bi3+ release of BiOCl under simulated physiological conditions in vitro, it is reasonable to infer that BiOCl retains considerable labeling efficiency even during later stages of degradation (Fig. S11). Considering that the intraoperative and early postoperative periods represent high-risk windows for embolus detachment when using BioGlue, the demonstrated in vivo labeling efficiency of Bi-BioGlue during the early stage ensures the reliable detection of emboli within this critical timeframe.

In Vivo Hemostasis and Healing on Rat Liver
After confirming their adhesive property and in vivo CT imaging capacity, Bi-BioGlue was applied to hemostasis in a liver puncture rat model (Fig. 3e). Hemorrhagic liver injury model was established by surgical creation of a wound 10 mm long and 3 mm deep. Subsequently, BioGlue and Bi-BioGlue were employed to treat the bleeding wound, and the untreated group set as a control. Both BioGlue and Bi-BioGlue achieved wound closure and hemostasis within 30 s, whereas severe blood loss occurred in the control group without any adhesive application (Fig. 3f). The amount of bleeding in control group, BioGlue group, and Bi-BioGlue group throughout the hepatic hemostasis process was measured to be 300 mg, 70 mg, and 65 mg, respectively. Bi-BioGlue exhibited a hemostatic effect similar to BioGlue, significantly reducing blood loss by 78.4% compared to the control group, thereby demonstrating the excellent hemostatic capability of Bi-BioGlue (Fig. 3g). The attachment of Bi-BioGlue to the liver was also proved by HE analysis after 24 h (Fig. 3h). Furthermore, the location and morphology of Bi-BioGlue were monitored by CT imaging, revealing gradual degradation over 42 days, consistent with pathological finding (Fig. 3i, j).

In Vitro CT Imaging of Bi-BioGlue
The detection limit of Bi-BioGlue was investigated for emboli of different sizes embedded within red meat of varying thicknesses (Fig. S12a). Ex vivo CT imaging showed that emboli with diameters of 1.0 mm (edge length of cubic emboli) and 1.2 mm. exhibited CT values of approximately 300 HU and 500 HU, respectively. These values were slightly lower than those of bulk samples due to partial volume effects. Notably, the CT values of Bi-BioGlue emboli were not significantly affected by the presence of varying thicknesses (1.0, 3.0, 5.0 cm) of overlying red meat, demonstrating the excellent tissue penetration capability of CT imaging.

Furthermore, we explored the influence of CT scanning parameters on embolus detectability, with particular attention to tube voltage and slice thickness. To achieve optimal visualization of ultra-small emboli, the slice thickness was set to the minimum value supported by the instrument (0.5 mm). When the tube voltage was varied between 80 and 140 kV, only minor fluctuations in CT values were observed across different embolus sizes, which can be attributed to the high K-edge energy of Bi (Fig. S12b, corresponding to Fig. 4a). These results indicated that changes in these scanning parameters did not significantly affect the embolus detection capability.

In Vivo CT Imaging of Bi-BioGlue-induced Pulmonary Embolism
To assess the feasibility of CT visualization for early and precise detection of adhesion emboli formed by surgical adhesives in vivo, an adhesive-induced pulmonary embolism model was established by puncturing and injecting a Bi-BioGlue embolus into the posterior vena cava, followed by sealing the puncture sites with Bi-BioGlue. Once entering the bloodstream, the emboli would flow with blood into pulmonary circulation and rapidly occlude the pulmonary artery within seconds or minutes (Fig. 4b). Then in vivo pulmonary embolism experiment was carried out according to the timeline of Fig. 4c. After modeling, the CT scan showed the location and pattern of Bi-BioGlue at posterior vena cava (Fig. 4d), meanwhile, a small bright nodule was observed obviously at middle area of right lung. Subsequently, spectral CT angiography (CTA) was performed through intravenous injection of iodine-based contrast agent (Iohexol). The CT images under mixed X-ray energies showed a visibly dilated pulmonary artery and abruptly interrupted in the middle area of right lung, indicating the occurrence of right pulmonary embolism, with a bright nodular signal at the occlusion site corresponding to the Bi-BioGlue embolus. (Fig. 4e). Compared to ex vivo conditions, the CT values of 1.0 mm and 1.2 mm Bi-BioGlue emboli decreased to approximately 220 HU and 400 HU, respectively, in vivo (Fig. S13). Owing to its small size and a CT value only slightly higher than that of surrounding tissues, the 1.0 mm embolus was difficult to reliably identify in vivo. In contrast, the 1.2 mm embolus exhibited markedly enhanced contrast against background tissue and was clearly visualized. Therefore, 1.2 mm was determined to be the minimum detectable embolus size in vivo using Bi-BioGlue.

Besides, monochromatic X-ray energy images of Spectral CTA were further constructed to elucidate the relationship between the abnormal nodule and the dilated artery. As Bi element possesses a higher K-edge value (91 keV) compared to iodine (33.2 keV), Bi-BioGlue exhibited strong X-ray attenuation in both low and high monochromatic X-ray energies, whereas Iohexol showed weak X-ray attenuation at high monochromatic X-ray energy (Fig. S14)56,57. Therefore, Bi-BioGlue could be distinguished from Iohexol using spectral CT. At low monochromatic X-ray image of 70 keV, both the dilated artery and abnormal nodule displayed high signals and blended. When increasing the monochromatic X-ray energy, the signal of dilated artery was sharply declined and could not even be clearly displayed at 130 and 160 keV, while the nodule was still visible and located at the end of dilated artery, demonstrating the nodule was embolized into the pulmonary artery and resulting in dilation of upstream vessels (Fig. 4f and Fig. S15). Additionally, HE analysis verified the pulmonary embolism induced by Bi-BioGlue (Fig. 4g). These experiments proved that incorporation of BiOCl in adhesive enables early and accurate diagnosis of adhesive embolism complication of BioGlue by CT imaging. Therefore, our approach enhanced BioGlue’s safety by addressing the clinical challenge of undetectable embolic complications, rather than altering its chemical composition. By enabling reliable post-operative monitoring of emboli, Bi-BioGlue provided a more comprehensive and proactive safety profile.

In Vitro Spectral CT Imaging for Differentiation of Bi-BioGlue-induced Embolus from Confusable Calcified Nodule
To further substantiate the clinical utility of Bi-BioGlue, we conducted in-depth study into the detection capability of emboli formed by Bi-BioGlue under complex background interference. In CT imaging, images of target areas are often affected by intracorporeal calcifications, such as vascular calcified plaques or pulmonary calcified nodules58–60. Therefore, distinguishing emboli caused by detachment of Bi-BioGlue from these calcifications presents a significant challenge. Considering that calcified plaques and nodules are mainly formed by organic material and calcium salt deposition, we utilized Ca3(PO4)2-doped BioGlue to simulate calcification nodules for in vitro and in vivo studies. To assess the specificity of spectral CT imaging in identifying Bi-BioGlue embolus in vitro, I-BioGlue, Bi-BioGlue, BioGlue injected into a well at row E and column 3, 6, 9 of 96-well plate, respectively, while filling the other remaining wells with Ca3(PO4)2-doped BioGlue (Fig. 5a). It was not surprisingly that the Bi-BioGlue was difficult to be distinguished from I-BioGlue, Ca-BioGlue in conventional CT (Fig. 5b). CT imaging results showed that Bi-BioGlue consistently maintained a high brightness across the entire range of X-ray energies, whereas BioGlue alone remained undetectable at all energy levels. Both I-BioGlue and Ca-BioGlue exhibited strong brightness at low X-ray energies, but their signals declined sharply as the X-ray energy increased, becoming extremely weak at higher energies (Fig. 5c, d). These findings clearly demonstrated that Bi-BioGlue enabled effective differentiation between emboli and calcified tissues in spectral CT imaging. In spectral CT, both Bi-BioGlue and Ca-BioGlue were clearly visible and indistinguishable from each other at low monochromatic X-ray energies of 40 and 70 keV. With the increase in monochromatic X-ray energy, the brightness of Ca-BioGlue’s CT images began to decrease and became almost invisible above 130 keV, whereas Bi-BioGlue maintained a relatively high contrast enhancement and could be clearly identified in the 96-well plate.

In Vivo Spectral CT Imaging for Differentiation of Bi-BioGlue-induced Embolus from Confusable Calcified Nodule
Subsequently, a rat model of pulmonary adhesive embolization concurrent with a calcified nodule was established by injecting a Bi-BioGlue embolus from the posterior vena cava and introducing a calcified nodule via intercostal space puncture. At low monochromatic X-ray energies of 40 and 70 keV, bone structures (such as ribs), Bi-BioGlue, and calcified nodules all exhibited pronounced bright effects on both sagittal and transverse CT images (Fig. 5e, f). Particularly, Bi-BioGlue emboli and calcified nodules showed similar morphology, signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) (SNR: 5.22 vs 5.28; CNR: 73.41 vs 60.92) (Fig. 5g, h). This similarity led to confusion and difficulties in detecting adhesive emboli. The emboli formed by Bi-BioGlue maintained a relatively high contrast enhancement; however, the enhancement effect of calcified nodules sharply declined. At 190 keV, the SNR of Bi-BioGlue was 4.05 times higher than that of the calcified nodule (Fig. 5g), demonstrating the superior contrast of Bi-BioGlue emboli compared to calcified nodules. These results demonstrated that even in the presence of complex background interference, Bi-BioGlue emboli can still be accurately detected using spectral CT imaging.

Biosafety Evaluation of Bi-BioGlue
The in vitro cytotoxicity was investigated by standard MTT assay. H9c2 cells and HUVEC, two of the most commonly used cell lines in cardiovascular research were cultured in media containing various concentrations of Bi-BioGlue for 24 and 48 h. The cell viabilities of H9c2 cell and HUVEC lines were remained higher than 80% even at a concentration as high as 20 mg/mL Bi-BioGlue which results suggested low cytotoxicity of Bi-BioGlue towards H9c2 cells and HUVEC (Fig. S16a, S16b).

For in vivo toxicology study, the rats were injected with Bi-BioGlue near the posterior vena cava, and then body weight monitoring, biochemical index analysis and hematoxylin and eosin (H&E) staining were carried out. During the 45 days’ monitoring, there was no significant body weights change between experimental group and control group (Fig. S16c). At days 1, 15, and 45 post-operation, the biochemical indexes (liver function markers: alanine aminotransferase, ALT; aspartate aminotransferase, AST; kidney function markers: urea, UREA; creatinine, CREA) and hematological parameters (white blood cell, WBC; red blood cell count, RBC; hemoglobin concentration, HGB; platelet count, PLT) of the experimental group rats were comparable to those of the control group (Fig. S16d, S16e). Moreover, the H&E slice images of major organs including heart, liver, spleen, lung and kidney showed no evident lesion, necrosis, and inflammation (Fig. S16f). The systematic toxicological assessment demonstrated Bi-BioGlue did not alter the intrinsic biosafety profile of the original BioGlue in term of chemical composition.

Methods
Materials
All chemicals used were of at least analytical grade. BSA, GA, Bi2S3, Bi2O3, BiCl3 and methylthiazolyltetrazolium (MTT) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) and ethanol were purchased from Concord Technology (Tianjin, China). Polyformaldehyde was obtained from Service Bio-Biotechnology Co., Ltd. (Wuhan, China). Ultra-pure water (Hangzhou Wahaha Group Co., Ltd.) was used throughout the synthesis process.

Synthesis of BiOCl and Bi-DTPA
In a typical produce, BiCl3 (1.5 mmol) was dissolved in 10 mL of deionized water, followed by ultrasonication for 10 min. The products were purified by centrifugation (424×g, 2 min) with ultrapure water three times.

The Bi-DTPA was fabricated according to our group reported method46. Briefly, Bi2O3 (0.5 mmol) and DTPA (1 mmol) were mixed in 20 mL H2O and stirred vigorously at 85°C for 2 h. The dark yellow suspension gradually became clear, indicating Bi-DTPA formation (pH=1.5). The pH was then adjusted to 7.4 with NaOH, and the Bi-DTPA product was freeze-dried for further use.

Synthesis of BioGlue, Bi-based BioGlue and I-doped BioGlue
The BioGlue used in this study was synthesized according to commercialized BioGlue product, which is a medical surgical adhesive developed and marketed by the American company CryoLife in the 1990s. It is a two-component adhesive, consisting of a 45% BSA solution and a 10% glutaraldehyde solution mixed in a 4:1 volume ratio.

To synthesize of Bi-based BioGlue, BiOCl, Bi-DTPA, Bi2S3 or Bi2O3 was first mixed and stirred with BSA solution to get Bi probe/BSA mixture with concentrations of Bi element at 22–131 mM and BSA at 450 mg/mL, followed by the addition of glutaraldehyde in a volume ratio of 4:1 to form Bi-based BioGlue. I-doped BioGlue was synthesized through replacing the Bi probe with Iohexol (22–131 mM) and other steps were the same as those of Bi-based BioGlue synthesis.

Synthesis of Hf-, Ta-, Yb-doped BioGlue
To synthesize Hf-, Ta-, and Yb-doped BioGlue, HfCl4, TaCl5, and YbCl3 were individually mixed and stirred with BSA solution to obtain metal probe/BSA mixtures containing 131 mM of the metal element and 450 mg/mL of BSA, respectively. Glutaraldehyde was then added at a volume ratio of 4:1 to form the corresponding Hf-, Ta-, and Yb-doped BioGlue.

Characterization
Transmission electron microscopy (TEM) (HT7700, Japan) was employed to characterize the morphology of BiOCl and Bi-DTPA. The morphology of Bi-BioGlue was characterized using FEI scanning electron microscopy (SEM) (Axio-Imager_LSM-800, German). Fourier transform infrared spectra (400-4000 cm-1) of BSA, GA, and Bi-BioGlue adhesives were recorded with a Nicolet iS10 spectrometer (Nicolet, USA), utilizing pure KBr as the background material. The X-ray diffraction (XRD) analysis was conducted using an Ultima IV X-ray diffractometer (Rigaku, Japan). Rheology experiments of BioGlue, Bi-, Hf-, Ta-, Yb-BioGlue were tested on an MCR xx2 rheometer (Anton Paar, Austria).

Stability Evaluation
To evaluate the morphological stability of Bi-BioGlue, a Bi-BioGlue cube with size of 5×5×5 mm was immersed in different media, including phosphate buffer solution (PBS, pH=7.4), normal saline, and Dulbecco’s modified eagle medium (DMEM, Gibco, USA). The stability of these samples was monitored by taking photos daily for 14 days.

To study the CT imaging stability of Bi-BioGlue, a Bi-BioGlue cube measuring 5×5×5 mm was immersed in ultrapure water, and changes in CT values were monitored via CT scans at various time intervals (1, 2, 4, 8, 12, and 24 h). For comparison, Bi-DTPA-BioGlue was also evaluated under the same conditions as those for Bi-BioGlue.

Both the Bi-BioGlue cube (5×5×5 mm) and BiOCl with equivalent Bi content were incubated in four representative media: H2O, physiological buffer (10 mM PBS, pH 7.4), an oxidative stress environment (1 mM H2O2), and serum. After different incubation periods (1, 7, and 14 days), the released Bi ions were quantified using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) analysis. Subsequently, the samples were freeze-dried for XRD characterization

Tensile Test
The tensile test is a widely used experimental method for evaluating the strength and adhesive properties of materials. Two clean porcine aortic blood vessels (each measuring 1×2 cm) were bonded using either Bi-BioGlue or BioGlue, with a bonded area of 1 cm2. After bonding, tensile tests were performed utilizing a mechanical tester to measure the force applied until the bonded vessels ruptured. The maximum pulling force was recorded to assess the adhesive capability of Bi-BioGlue.

Ex Vivo Tissue Adhesion of Bi-BioGlue
To evaluate the tissue adhesive performance of Bi-BioGlue, an ex vivo leakage model was established using isolated pig aorta vessels. A longitudinal incision was made on the vessel wall to simulate a leakage site. Bi-BioGlue was used to the treatment of the defect using a syringe, followed by 30 s of contact to initiate adhesion. The repaired vessel was then allowed to stand undisturbed for 120 s to ensure complete gelation and bonding. Subsequently, red ink was injected from the proximal end of the vessel to simulate blood flow. The absence of ink leakage from the repair site was used as an indicator of effective tissue adhesion.

Cytotoxicity
Rat cardiomyocytes (H9c2) cells and HUVEC were purchased from Cell Bank of Chinese Academy of Sciences and used to investigate the cytocompatibility of Bi-BioGlue in vitro. Firstly, the freeze-dried Bi-BioGlue were soaked in the culture medium with different concentrations (0, 1, 3, 5, 10, 15, 20 mg/mL) and incubated for 24 h at 37°C to obtain the Bi-BioGlue extracts. H9c2 cells and HUVEC were cultured in 96-well plates with a density of 1×104 per well for 24 h at 37°C with 5% CO2. Afterwards, the cells were rinsed with phosphate buffer solution (PBS, 10 mM, pH=7.4) and Bi-BioGlue extracts was added to the wells and incubated for another 24 h or 48 h. Subsequently, H9c2 cells and HUVEC were rinsed with phosphate buffer solution and fresh culture medium supplemented with MTT (5 mg/mL, 10 μL) was added to the cells and incubated for 4 h at 37°C with 5% CO2. The MTT-containing medium was then replaced, and DMSO (120 μL) was added to dissolve the blue-purple formazan crystals formed at the bottom of the wells. After 10 min of gentle shaking, 490 nm absorption value of each well were measured using a microwell plate reader (Bio-Tek, USA). The cell viability was defined and calculated as follows: Cell viability = ODexp / ODcon × 100%, of which ODexp is the optical density (OD) of cells cocultured with diverse concentrations of Bi-BioGlue adhesive, and ODcon means that of the control group.

In Vivo Toxicity Study
All animal experiments were performed in compliance with the guidelines of the Animal Care and Use Committee of Tianjin Medical University General Hospital, and approved by the Animal Care and Use Committee of Tianjin Medical University General Hospital (IRB 2022-DW-76). Sprague-Dawley (SD) rats (280-320 g) were provided by SPF Biotechnology Co., Ltd. (Beijing, China). Animals were bred and housed under a 12:12 h light-dark cycle at an ambient temperature of 22°C and a relative humidity ranging from 40 to 70%.

Sixteen male SD rats were randomly divided into four groups (Control group, 1-, 15- and 45-day groups), with four rats in each group. Rats in 1-, 15- and 45-day groups were anesthetized with isoflurane and underwent a midline abdominal incision to expose the posterior vena cava for injection of 30 μL of Bi-BioGlue, followed by suturing the incisions. Rats in control group underwent a similar surgical procedure without receiving Bi-BioGlue. The body weight changes of rats in control group and 45-day group were recorded every three days. After 1, 15, and 45 days post-operation, the rats in the corresponding group were sacrificed, and the rats in Control group were sacrificed after 45 days post-operation. Major organs including heart, liver, spleen, lung and kidney were fixed in paraformaldehyde for histopathological analysis. Blood samples were collected for biochemical analysis and routine blood tests.

In Vivo Monitoring of Bi-BioGlue via CT Imaging
SD rats (n=3) were anesthetized with isoflurane and placed in supine position for abdominal fur removal. A 2 cm incision was made along the midline of the abdomen to expose the posterior vena cava. Bi-BioGlue was injected to the posterior vena cava, followed by free the clips and suturing the abdominal incision. After that, spectral CT scanning was performed on a clinical CT scanner (Somatom Drive, Siemens Healthineers, Erlangen, Germany) to monitor the changes of Bi-BioGlue colloid at 1, 7, 28 and 42 days post-operation under following scanning parameters: field of view (FOV) was 256×256 mm, slice thickness was 0.5 mm, with adaptive tube current and voltage were set at 80/140 Sn.

In Vivo Hemostasis and Healing on Rat Livers
SD rats were divided into three groups (n=3 for each group), including control group, Bi-BioGlue group and BioGlue group. The rats (n=3 for each group) were anesthetized with isoflurane and placed in supine position for abdominal fur removal. A 10 mm lateral incision was made to expose the liver, followed by placing a filter paper under the liver. A 10 mm long, 3 mm deep incision was made on the left hepatic lobe with a surgical scalpel. After inducing liver bleeding, rats in the Bi-BioGlue and BioGlue groups were treated with 60 μL of Bi-BioGlue or BioGlue, respectively. Both adhesives sealed the bleeding site and stopped hemorrhage within approximately 30 s. Rats in the control group did not receive any treatment and were allowed to bleed and coagulate naturally. The liver was returned to its original position in the abdomen and the incision was sutured. The filter paper was weighted before and after operation to quantify the amount of blood loss. Spectral CT scan was carried out before and after surgery (1, 3, 7, 14, 21, 28, and 42 days post-operation). scanning parameters: FOV was 256 × 256 mm, slice thickness was 0.5 mm, with adaptive tube current and voltage were set at 80/140 Sn.

SD rats were divided into three groups (n=3 for each group) randomly and treated as mentioned above. At 24 h post-operation, the rats were euthanized, and the liver were harvested for histopathological analysis of operation site.

Ex Vivo CT imaging of Bi-BioGlue Emboli with Different Parameters
Bi-BioGlue was carefully trimmed into cubic emboli with diameters of 1.0, 1.2, 2.0, 3.0, 4.0, and 5.0 mm. CT imaging was conducted under various conditions to evaluate the detectability of emboli in ex vivo settings.

Scanning parameters optimization: emboli were imaged while embedded in red meat. Slice thicknesses were varied at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0 mm, and the tube voltage was adjusted between 80, 100, 120, and 140 kV.

Impact of penetration depth on the detectability of Bi-BioGlue emboli: To assess the penetration capability of CT, 1.2 mm emboli were sandwiched between red meat slices of 1.0, 3.0, and 5.0 cm thickness. CT scanning was conducted with a FOV of 256×256 mm, slice thickness of 0.5 mm, and tube voltage of 120 kV.

Detection limits in air and tissue: emboli were placed either in air or sandwiched between two 2 cm-thick slices of red meat. Scanning was performed with a FOV of 256×256 mm, a slice thickness of 0.5 mm, and adaptive tube current at 120 kV.

In Vivo CT Visualization of Bi-BioGlue-induced Pulmonary Embolism
SD rats (n=3) were anesthetized with isoflurane and placed in a supine position for abdominal fur removal. A 2 cm incision was made along the median line of the abdomen to expose the inferior vena cava. The lower end of posterior vena cava, bilateral renal vein and common iliac veins were clamped with vascular clips. A cubic Bi-BioGlue colloid with size of 1.2 mm was injected into the posterior vena cava through a puncture needle. The upper end of posterior vena cava was clamped with vascular clip. After withdrawal of the needle, the puncture site was adhered with Bi-BioGlue, followed by free all clips and suturing the abdominal incision. Before and after operation, spectral CT scanning was performed to visualize the Bi-BioGlue and search for embolus under following scanning parameters: the FOV was set to 256×256 mm with a slice thickness of 0.5 mm. The adaptive tube current and voltage were 80/140 Sn. SNR and CNR were calculated according to following formula: SNR=CT value of ROI/SD of background, CNR = (CT value of ROI - CT value of muscle)/SD of background. Subsequently, Iohexol enhanced spectral CT was performed to verify the occurrence of embolism under following scanning parameters: the FOV was set to 256 × 256 mm with a slice thickness of 0.5 mm. The adaptive tube current and voltage were 80/140 Sn. Spectral images at various monochromatic energies (70-190 keV) were processed using the syngo.via viewer (Siemens Healthineers, Germany). Finally, the rats were euthanized, and histopathological analysis was conducted on the excised pulmonary lobes containing the embolus.

In Vitro Differentiation of Bi-BioGlue and Calcified Nodule through CT Imaging
To prepare Ca3(PO4)2-doped BioGlue, 4.5 g of Ca3(PO4)2 and 4 g of BSA are weighed and dissolved in water to form Ca3(PO4)2/BSA mixture (final volume 10 mL, Ca3(PO4)2 1.45 mol/L, BSA 400 mg/mL), followed by stirring evenly. It is worth noting that during the preparation of Ca3(PO4)2-doped BioGlue, the concentration of BSA used was 400 mg/mL rather than 450 mg/mL. This adjustment was made because a higher BSA concentration, combined with calcium phosphate, is difficult to uniformly disperse in water. Since the CT contrast primarily arises from the calcium content, the slight reduction in BSA concentration is not expected to affect the experimental outcomes. Next, Ca3(PO4)2/BSA-doped BioGlue was formed by reacting a mixture of Ca3(PO4)2/BSA and 10% glutaraldehyde in a 96-well plate, with volumes of 160 μL/well and 40 μL/well, respectively. In parallel, 200 μL of I-BioGlue, Bi-BioGlue, and BioGlue were added to wells in column E, rows 3, 6, and 9, respectively. The concentrations of iodine and bismuth were adjusted to 105 mM in I-BioGlue and Bi-BioGlue, respectively. The remaining wells were filled with Ca-BioGlue. Subsequently, both conventional CT scanning and spectral CT scanning were carried out following scanning parameters: the FOV was set to 256×256 mm with a slice thickness of 0.5 mm. The adaptive tube current and voltage were 80/140 Sn, equivalent to a conventional 120 kV. Spectral images at various monochromatic X-ray energies (40-130 keV) were processed using the syngo.via viewer (Siemens Healthineers, Germany).

In Vivo Differentiation of Bi-BioGlue-induced Pulmonary Embolism and Calcified Nodule through CT Imaging
In this experiment, Ca3(PO4)2 doped BioGlue (Ca concentration of 1.16 M) were utilized to simulate calcified nodule. SD rats (n=3) were anesthetized with isoflurane and placed in a supine position for chest and abdominal fur removal. A 2 cm incision was made along the median line of the abdomen to expose the inferior vena cava. The lower end of the renal vein and inferior vena cava, and the upper end of the left and right common iliac veins were clamped with vascular clips. Bi-BioGlue embolus with size of 1.2 mm was injected into the posterior vena cava through a puncture needle to cause pulmonary embolism. After withdrawal of the needle, the puncture site was adhered with Bi-BioGlue, followed by free the clips and suturing the abdominal incision. Then, calcified nodule stimulated with Ca3(PO4)2 doped BioGlue was punctured into the lung. After that, spectral CT scanning was performed to classify of calcified nodule and Bi-BioGlue-induced embolus. Scanning parameters: the FOV was set to 256×256 mm with a slice thickness of 0.5 mm. The adaptive tube current and voltage were 80/140 Sn, equivalent to a conventional 120 kV. Spectral images at various monochromatic X-ray energies (40-190 keV) were processed using the syngo.via viewer (Siemens Healthineers, Germany). SNR and CNR were calculated according to following formula: SNR=CT value of ROI/SD of background, CNR=(CT value of ROI - CT value of muscle)/SD of background.

Statistics and Reproducibility
The results in all the experiments were presented as mean ± standard deviation (s.d.). Statistical calculation of experimental data was analyzed using One-Way Analysis of Variance (ANOVA) and Two-Way ANOVA. Article drawing was performed using Origin 2019 or GraphPad Prism 8.0.2 software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors incorporated bismuth (Bi) element in the form of BiOCl into a commercial surgical adhesive (BioGlue) for\nnoninvasive detection of adhesive-induced emboli using spectral CT imaging, with detection sensitivity reportedly as small\nas 1.2 mm. However, despite these findings, the use of bismuth as a CT contrast agent has been extensively documented,\nand BioGlue, as a commercial surgical adhesive, does not present significant novelty. More importantly, the work lacks\nsufficient evidence to fully support its claims and conclusions. Therefore, I do not recommend this manuscript for further\nconsideration in Nature Communications. The manuscript can be strengthened by addressing the following points:\n1. The authors mention that the CT value of Bi-DTPA-BioGlue decreased within 5 hours when soaked in water, attributing\nthis to the small molecular size and good water solubility of Bi-DTPA. However, how does BiOCl compare? Does BiOCl\nhave a sufficiently large molecular size or poor water solubility to ensure its retention in BioGlue? Clarification on this point\nis needed.\n2. The authors conclude that the CT imaging detection limit for Bi-BioGlue emboli is 1.2 mm. However, this conclusion\nraises several concerns: (1) In Figure 4a, the 1 mm embolus in red meat is still visible and distinguishable from the\nsurrounding background, calling the 1.2 mm detection limit into question. (2) The authors should verify whether factors such\nas the amount of Bi doped in BioGlue, the depth of the Bi-BioGlue within the tissue, and the CT machine parameters could\ninfluence the detection limit. (3) Additionally, the authors should consider employing a more standardized and scientific\nmethod to confirm the detection limit, rather than relying solely on visual judgment.\n3. The authors used spectral CT to differentiate Bi-BioGlue from Ca-BioGlue. However, two additional control groups should\nbe included for comparison: (1) BioGlue and Ca-BioGlue, and (2) Iodine-BioGlue and Ca-BioGlue. These controls would\nbetter demonstrate that only the developed Bi-BioGlue can effectively distinguish emboli from calcified tissues.\n4. In Figures 5e and 5f, the authors should clarify what the two types of arrows represent.\n5. The flow of results is disjointed. The authors first discuss Figure 4a, then move to Figure 5a-d, before returning to Figure\n4b-g, and finally return to Figure 5e-h. This organization is confusing and should be reconsidered for better coherence.\n6. The authors should provide more information on how long it takes for BioGlue to form a pulmonary embolism in vivo.\n7. The rationale for using H9C2 cells, a cardiomyocyte cell line, for the biosafety evaluation should be explained, as this cell\nline may not be the most appropriate model for assessing BioGlue’s general biocompatibility.", "reviewer_2": "The manuscript describes the characterization of a bismuth labelled derivative of a clinically used surgical adhesive, and\npreclinical in vivo assessment of the feasibility to visualize vascular emboli using this compound using CT imaging. The\nauthors show that the bismuth labelled derivative has the same adhesive capabilities compared to the FDA approved Bio-\nGlue.\nMajor issues that need to be addressed to improve the value of the manuscript:\n- In the introduction the size of emboli when damage occurs needs to be mentioned and related to the size detected in the\nresults. The authors also need to explain the benefit of hypersensitive detection of millimetre sized emboli. How do the\nauthors envision clinical application? Through watch and wait or intervention therapy?\nThe extent of symptoms will be dependent on the size of the embolism. Will there be differences in treatment compared to\nstandard embolisms? As anticoagulants will not work and nor will fibrinolytic therapy.\n- Line 48: Optical imaging is not a suitable imaging methodology for this application due to its limited penetration depth and\nshould be excluded here.\n- Line 53: The authors present the bismuth labelling as novel, but this has already been applied for adhesives. Tian et al\n(Advanced materials 2023) describe a bismuth-pectin organic-inorganic complex that was engineered into a transformable\nmicrogel (that they call bi-glue) and that is compatible with x-ray imaging. The statement made by the authors needs to be\nrevised and the paper by Tian et al should be accurately referenced and the differences in approaches should be discussed.\n- Fig 3 shows the visualization of emboli over the course of 42 days. What was the percentage of resorbance of emboli? If all\nwere resorbed, what is then the additional value of early detection?\n- An explanation should be provided to how detection of millimetre sized emboli will significantly improve the biosafety of\nsurgical adhesions. In my opinion these two features are not directly linked as the biosafety profile of the adhesive is\ndependent on the chemical composition itself and not on its detectability. Figure 6 shows biosafety assessment of B-BioGlue\nwithout comparison to the original BioGlue, therefore significant improvement cannot be claimed.\n- Stability assessment of the created complex is insufficient. How can the authors ensure that the complex will remain stable\nin vivo? This should at least be tested under comparable conditions.\nHow is the apparent degradation of the adhesive being taken into account? It needs to be investigated that the Bi remains\ncomplexed during degradation. The presence of non-labelled adhesive and a possible effect on emboli formation and\nvisualization needs to be assessed.", "reviewer_3": "The authors present a bismuth-based BiOCl-doped BioGlue for detecting vascular emboli originating from surgical\nadhesives using CT imaging. This CT-visible adhesive enables in vivo tracking of adhesive degradation and the real-time\ndetection of small emboli. The Bi-BioGlue demonstrates enhanced stability, biocompatibility, and prolonged imaging\npersistence, with the capability to detect emboli as small as 1.2 mm in a pulmonary embolism model. Overall, this study is\nwell-designed with a rigorous methodology. However, several questions and suggestions need to be addressed before\nconsidering publication.\n1. The authors used known molecules to form a crosslinked glue. However, the chemistry should be shown more clearly,\nincluding the chemical structures of each molecule and the detailed characterizations after synthesis.\n2. The cross-linking mechanism between BiOCl and the BioGlue matrix is briefly mentioned, but more experimental details\nare required. This should include the kinetics and degree of cross-linking, as these factors could directly influence adhesion\nstrength and stability. Additionally, as BiOCl stability is crucial for sustained imaging, please discuss the stability of BiOCl in\nthe adhesive under physiological pH and oxidative conditions.\n3. On page 6, line 105, please include a reference for the synthesis method of Bi-DTPA.\n4. In Fig S2, please add or label BioGlue, gelatin, and sodium alginate within the figures.\n5. In Fig. 2g, please clarify what the colors represent.\n6. In Fig. 2k, what are the concentrations of Bi-BioGlue and iohexol-doped BioGlue used in this study? If the concentration is\n105 mM, please explain the rationale for this choice. Additionally, please provide a more detailed comparison of detection\nsensitivity with standard iodine-based agents.\n7. In Fig. S3, the color change in DMEM from D0 to D14 (red to yellow) likely reflects pH changes. Please provide an\nexplanation of any chemical reactions or degradation processes that may have occurred during the time period.\n8. On page 7, line 141, verify if Fig. 1c is correctly referenced.\n9. In Fig. S6, please provide experimental details, including bonding duration and the time elapsed before testing Bi-\nBioGlue.\n10. In Fig. 3f, indicate the time required to achieve sealing and hemostatic effects. This information should be included in the\nexperimental section or figure legend.\n11. In Fig. 4a, provide more details regarding the preparation of Bi-BioGlue emboli in air and in red meat.\n12. In Fig. 5a, describe the process for creating Ca3(PO4)2-doped BioGlue.\n13. On page 15, line 317, ensure consistency in units, as “1.2 mm” appears in several places. Please clarify if this\nmeasurement refers to diameter or volume.\n14. On pages 9 and 12, where descriptions for Fig. 4 and Fig. 5 are located, reorganize the paragraphs to improve flow and\nreadability.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Response to Reviewers’ Comments\nThe comments and suggestions made by the reviewers are very helpful for us to\nrevise the manuscript, and we highly appreciate the reviewers for such constructive\ncomments. Detailed reply to the questions and concerns is made below:\nResponse to Reviewer 1\nComments:\nThe authors incorporated bismuth (Bi) element in the form of BiOCl into a\ncommercial surgical adhesive (BioGlue) for noninvasive detection of\nadhesive-induced emboli using spectral CT imaging, with detection sensitivity\nreportedly as small as 1.2 mm. However, despite these findings, the use of bismuth as\na CT contrast agent has been extensively documented, and BioGlue, as a commercial\nsurgical adhesive, does not present significant novelty. More importantly, the work\nlacks sufficient evidence to fully support its claims and conclusions. Therefore, I do\nnot recommend this manuscript for further consideration in Nature Communications.\nThe manuscript can be strengthened by addressing the following points:\nReply:\nThank you very much for the careful review and valuable comments on our\nmanuscript. We respectfully believe that there may be a misunderstanding regarding\nthe objectives and contributions of our work, and we would like to take this\nopportunity to clarify these aspects.\nFirst, regarding the issue of novelty, you noted that Bi has been widely used for\nCT imaging and that BioGlue is a commercially available surgical adhesive. However,\nthe novelty of our work does not lie in the development of a new adhesive, but rather\nin establishing a sensitive and practical CT imaging strategy to detect emboli formed\nby the detachment of BioGlue, an unmet clinical need. While Bi-based probes have\nbeen explored for imaging applications, their integration with adhesive systems for\nprecise embolus detection requires careful material design and optimization. In our\nstudy, we systematically screened a range of Bi-based candidates and identified\namorphous BiOCl as a uniquely suitable imaging probe for BioGlue labeling. This\nrepresents a novel and rationally engineered solution tailored to the clinical challenge\nof embolus visualization.\nOur work, therefore, introduces a previously unexplored CT imaging approach\nfor tracking BioGlue-derived emboli in vivo with high precision and sensitivity. We\nbelieve this contribution offers not only materials innovation and methodological\nadvances, but also substantial clinical relevance.\nSecond, in response to the concerns regarding data support, we would like to\nemphasize that the study is grounded in a comprehensive and rigorous experimental\nframework. This includes rational probe design, detailed physicochemical\ncharacterization, and well-validated in vivo imaging studies. As acknowledged by\nReviewer 3, “Overall, this study is well-designed with a rigorous methodology”. We\nhave also conducted additional experiments to further strengthen the conclusions and\nhave revised the manuscript accordingly to address all technical comments. These\nexperiments cover the entire workflow, including material synthesis and\ncharacterization, as well as biological evaluation. Specifically, they include solubility\nand microstructure analysis of BiOCl, assessment of Bi-BioGlue’s stability under\nphysiological conditions, detection limit for emboli, spectral CT imaging performance,\nand comprehensive biosafety evaluation.\nWe sincerely thank you again for the rigorous review and constructive\nsuggestions. In the revised manuscript, we have carefully addressed all concerns, and\nthe combination of original and newly added data now provides strong and\ncomprehensive support for our conclusions. We respectfully invite you to re-evaluate\nour work in light of its demonstrated innovation and the improved robustness of the\nsupporting evidence.\nComment 1:\nThe authors mention that the CT value of Bi-DTPA-BioGlue decreased within 5\nhours when soaked in water, attributing this to the small molecular size and good\nwater solubility of Bi-DTPA. However, how does BiOCl compare? Does BiOCl have\na sufficiently large molecular size or poor water solubility to ensure its retention in\nBioGlue? Clarification on this point is needed.\nReply and corresponding changes:\nThank you very much for your valuable comment. As suggested by the reviewers,\nwe conducted additional experiments to compare the particle size and aqueous\nsolubility of BiOCl and Bi-DTPA. The results showed that Bi-DTPA readily formed a\nhighly transparent solution in water, confirming its excellent solubility (Fig. S1a). In\nstark contrast, BiOCl quickly formed flocculent precipitates upon dispersion,\nindicating significantly lower water solubility than Bi-DTPA (Fig. S1b). Additionally,\nBi-DTPA, being a small molecular compound with high solubility, is difficult to\nvisualize under TEM (Fig. S1c). In contrast, TEM images revealed that BiOCl\nparticles were composed of aggregated nanoparticles, a morphology consistent with\ntheir poor solubility (Fig. S1d). Consequently, BiOCl exhibited a much slower release\nrate from the BioGlue matrix, with the CT signal of BiOCl-BioGlue remaining stable\nfor over 24 h. By comparison, the CT value of Bi-DTPA-BioGlue decreased markedly\nwithin 5 h, due to the rapid diffusion of Bi-DTPA from the hydrogel.\nRevised text\n…BiOCl, composed of aggregated nanoparticles with poor water solubility, was\nfabricated using a simple hydrolysis method (Fig. S1d, e), and Bi O and Bi S were\n2 3 2 3\nobtained through commercial sources. (Page 5)\nRevised figure\nFig. S1. Photographs of Bi-DTPA (a) and BiOCl (b) dispersions in water under static\nconditions. TEM image of Bi-DTPA (c) and BiOCl (d). (e) XRD pattern of BiOCl.\nComment 2:\nThe authors conclude that the CT imaging detection limit for Bi-BioGlue emboli\nis 1.2 mm. However, this conclusion raises several concerns: (1) In Figure 4a, the 1\nmm embolus in red meat is still visible and distinguishable from the surrounding\nbackground, calling the 1.2 mm detection limit into question. (2) The authors should\nverify whether factors such as the amount of Bi doped in BioGlue, the depth of the\nBi-BioGlue within the tissue, and the CT machine parameters could influence the\ndetection limit. (3) Additionally, the authors should consider employing a more\nstandardized and scientific method to confirm the detection limit, rather than relying\nsolely on visual judgment.\nReply and corresponding changes:\nThank you very much for your valuable comments! As the reviewer suggested,\nwe have revised and provided a detailed and specific description of the emboli\ndetection limit, and systematically investigated the impacts of Bi doping\nconcentration, embolus embedding depth, and key CT machine parameters on the\nembolus detection capability in the scanning results.\nWe systematically optimized the Bi doping concentration to assess its effect on\nthe CT imaging performance of Bi-BioGlue. Increasing the Bi content enhanced the\nsensitivity of CT detection; however, excessive doping led to undesirable changes in\nthe colloidal properties and raised potential concerns about biosafety. Our results\nshowed that the CT value of bulk Bi-BioGlue gradually increased with rising Bi\nconcentrations. When the Bi concentration reached 105 mM, the CT value of\nBi-BioGlue reached approximately 659 HU, yielding a favorable\nsignal-to-background ratio compared to normal tissues, which typically exhibit CT\nvalues in the range of 50-80 HU. It should be noted that maintaining a high CT value\nin bulk Bi-BioGlue relative to soft tissue is essential, as the formation of very small\nBioGlue emboli will lead to a certain degree of CT signal attenuation due to partial\nvolume effects. Importantly, at this concentration, the mechanical and adhesive\nproperties of BioGlue were not adversely affected. Therefore, 105 mM was selected\nas the optimal Bi doping concentration for the following studies.\nWe also investigated the detection limit of Bi-BioGlue for emboli of different\nsizes embedded within red meat of varying thicknesses. Ex vivo CT imaging showed\nthat emboli with diameters of 1.0 mm and 1.2 mm exhibited CT values of\napproximately 300 HU and 500 HU, respectively. These values are slightly lower than\nthose of bulk samples due to partial volume effects. Notably, the CT values of\nBi-BioGlue emboli were not significantly affected by the presence of varying\nthicknesses (1.0, 3.0, 5.0 cm) of overlying red meat, which is attributed to the\nexcellent tissue penetration capability of CT imaging (Fig. S12a). In vivo, these\nvalues decreased to approximately 220 HU and 400 HU, respectively (Fig. S13). Due\nto its small size and a CT value only marginally higher than surrounding tissue, the\n1.0 mm embolus posed significant challenges for reliable detection in vivo. In contrast,\nthe 1.2 mm embolus exhibited a significantly stronger contrast against background\ntissues and could be clearly detected. Therefore, 1.2 mm was determined to be the\nminimum detectable embolus size in vivo using Bi-BioGlue.\nFurthermore, we explored the influence of CT scanning parameters on embolus\ndetectability, with particular attention to tube voltage and slice thickness. To achieve\noptimal visualization of ultra-small emboli, the slice thickness was set to the\nminimum value supported by the instrument (0.5 mm). When the tube voltage was\nvaried between 80 and 140 kV, only minor fluctuations in CT values were observed\nacross different embolus sizes, which can be attributed to the high K-edge energy of\nBi (Fig. S12b). These results indicate that changes in scanning parameters do not\nsignificantly affect the detection limit, and that 1.2 mm remains the smallest embolus\nsize that can be reliably detected under a range of CT imaging conditions.\nRevised text and figures\nBi doping concentration was systematically optimized to assess its effect on the\nCT imaging performance of Bi-BioGlue. Increasing the Bi content enhanced the\nsensitivity of CT detection; however, excessive doping led to undesirable changes in\nthe colloidal properties and raised potential concerns about biosafety. The result\nshowed that the CT value of bulk Bi-BioGlue gradually increased with rising Bi\nconcentrations. When the Bi concentration reached 105 mM, the CT value of\nBi-BioGlue reached approximately 659 HU, yielding a favorable\nsignal-to-background ratio compared to normal tissues, which typically exhibit CT\nvalues in the range of 50-80 HU. It should be noted that maintaining a high CT value\nin bulk Bi-BioGlue relative to soft tissue is essential, as the formation of very small\nBioGlue emboli will lead to a certain degree of CT signal attenuation due to partial\nvolume effects. Therefore, 105 mM was selected as the optimal Bi doping\nconcentration for the following studies…(Page 5)\nThe crosslinking kinetics of both BioGlue and Bi-BioGlue by rheological\nanalysis demonstrated that upon mixing, the BSA and glutaraldehyde components\nexhibited similarly rapid crosslinking kinetics, forming an opaque white solid within\nseconds (Fig. 2h) … (Page 6)\nFig. 2. (h) Dynamic oscillatory time sweep measurement of BioGlue and Bi-BioGlue.\n…The detection limit of Bi-BioGlue was investigated for emboli of different\nsizes embedded within red meat of varying thicknesses (Fig. S12a). Ex vivo CT\nimaging showed that emboli with diameters of 1.0 mm and 1.2 mm exhibited CT\nvalues of approximately 300 HU and 500 HU, respectively. These values are slightly\nlower than those of bulk samples due to partial volume effects. Notably, the CT values\nof Bi-BioGlue emboli were not significantly affected by the presence of varying\nthicknesses (1.0, 3.0, 5.0 cm) of overlying red meat, demonstrating the excellent\ntissue penetration capability of CT imaging.\nFurthermore, we explored the influence of CT scanning parameters on embolus\ndetectability, with particular attention to tube voltage and slice thickness. To achieve\noptimal visualization of ultra-small emboli, the slice thickness was set to the\nminimum value supported by the instrument (0.5 mm). When the tube voltage was\nvaried between 80 and 140 kV, only minor fluctuations in CT values were observed\nacross different embolus sizes, which can be attributed to the high K-edge energy of\nBi (Fig. S12b, corresponding to Fig. 4a). These results indicate that changes in\nscanning parameters do not significantly affect the embolus detection capability.\n(Page 8)\nFig. S12. (a) CT images of Bi-BioGlue emboli in red meat with different thicknesses\n(0, 1.0, 3.0, 5.0 cm). (b) CT images of Bi-BioGlue emboli of different sizes in red\nmeat under different voltages (80, 100, 120, 140 kV) and scanning layer thicknesses\n(0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0 mm).\nCompared to ex vivo conditions, the CT values of 1.0 mm and 1.2 mm\nBi-BioGlue emboli decreased to approximately 220 HU and 400 HU, respectively, in\nvivo (Fig. S13). Owing to its small size and a CT value only slightly higher than that\nof surrounding tissues, the 1.0 mm embolus was difficult to reliably identify in vivo.\nIn contrast, the 1.2 mm embolus exhibited markedly enhanced contrast against\nbackground tissue and was clearly visualized. Therefore, 1.2 mm was determined to\nbe the minimum detectable embolus size in vivo using Bi-BioGlue. (Page 10)\nFig. S13. CT images of 1.0 mm Bi-BioGlue emboli in vivo.\nEx Vivo CT imaging of Bi-BioGlue Emboli with Different Parameters.\nBi-BioGlue was carefully trimmed into cubic emboli with diameters of 1.0, 1.2,\n2.0, 3.0, 4.0, and 5.0 mm. CT imaging was conducted under various conditions to evaluate\nthe detectability of emboli in ex vivo settings.\nScanning parameters optimization: emboli were imaged while embedded in 2\ncm-thick red meat. Slice thicknesses were varied at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 10\nmm, and the tube voltage was adjusted between 80, 100, 120, and 140 kV.\nImpact of penetration depth on the detectability of Bi-BioGlue emboli: To assess\nthe penetration capability of CT, 1.2 mm emboli were sandwiched between red meat\nslices of 1.0, 3.0, and 5.0 cm thickness. CT scanning was conducted with a FOV of\n256 × 256 mm, slice thickness of 0.5 mm, and tube voltage of 120 kV.\nDetectionl limits in air and tissue: Emboli were placed either in air or\nsandwiched between two 2 cm-thick slices of red meat. Scanning was performed with\na FOV of 256 × 256 mm, a slice thickness of 0.5 mm, and adaptive tube current at\n120 kV. (Pages 15-16)\nComment 3:\nThe authors used spectral CT to differentiate Bi-BioGlue from Ca-BioGlue.\nHowever, two additional control groups should be included for comparison: (1)\nBioGlue and Ca-BioGlue, and (2) Iodine-BioGlue and Ca-BioGlue. These controls\nwould better demonstrate that only the developed Bi-BioGlue can effectively\ndistinguish emboli from calcified tissues.\nReply and corresponding changes:\nThank you very much! As the reviewers suggested, we have supplemented the\nscanning images of 96-well plate spectral CT at different keV for comparisons among\nCa-BioGlue, Iodine-BioGlue, and BioGlue, as depicted in the figure. 200 μL of\nI-BioGlue, Bi-BioGlue, and BioGlue were placed in positions E3, E6, and E9 of the\n96-well plate respectively, and Ca-BioGlue was added to the remaining holes. CT\nimaging results showed that Bi-BioGlue consistently maintained a high brightness\nacross the entire range of X-ray energies, whereas BioGlue alone remained\nundetectable at all energy levels. Both I-BioGlue and Ca-BioGlue exhibited strong\nbrightness at low X-ray energies, but their signals declined sharply as the X-ray\nenergy increased, becoming extremely weak at higher energies. These findings clearly\ndemonstrate that Bi-BioGlue enables effective differentiation between emboli and\ncalcified tissues in spectral CT imaging (Fig. S5a-d).\nRevised text\nI-BioGlue, Bi-BioGlue, BioGlue injected into a well at row E and column 3, 6, 9\nof 96-well plate, respectively, … CT imaging results showed that Bi-BioGlue\nconsistently maintained a high brightness across the entire range of X-ray energies,\nwhereas BioGlue alone remained undetectable at all energy levels. Both I-BioGlue\nand Ca-BioGlue exhibited strong brightness at low X-ray energies, but their signals\ndeclined sharply as the X-ray energy increased, becoming extremely weak at higher\nenergies (Fig. 5c, d). These findings clearly demonstrate that Bi-BioGlue enables\neffective differentiation between emboli and calcified tissues in spectral CT imaging.\n(Page 12)\nRevised figure\nFig. 5. Spectral CT imaging for differentiation of Bi-BioGlue-induced embolus from\nconfusable calcified nodule. (a) Schematic illustration of I-BioGlue injected into a\nwell at row E and column 3, Bi-BioGlue injected into a well at row E and column 6,\nBioGlue injected into a well at row E and column 9 of 96-well plate, while filling\nother wells with Ca (PO ) -doped BioGlue. Conventional CT images (b) and spectral\n3 4 2\nCT images (c) of 96-well plate. (d) Spectral CT signal curves of Bi-BioGlue,\nI-BioGlue, BioGlue and Ca (PO ) -doped BioGlue at different monochromatic\n3 4 2\nenergies. … (Page 11)\nComment 4:\nIn Figures 5e and 5f, the authors should clarify what the two types of arrows\nrepresent.\nReply and corresponding changes:\nThank you very much! We sincerely apologize for omitting the two types of\narrows represent in Figures 5e and 5f. A detailed description about the two types of\narrows have been provided in revised manuscript, which the thin arrows herein\nindicate Ca-BioGlue simulated pulmonary calcification, and the wide arrows signify a\npulmonary embolism caused by Bi-BioGlue.\nRevised text\nFig. 5. …The calcified nodule is indicated by a thin arrow, and the Bi-BioGlue\nembolus is indicated by a wide arrow… (Page 11)\nComment 5:\nThe flow of results is disjointed. The authors first discuss Figure 4a, then move\nto Figure 5a-d, before returning to Figure 4b-g, and finally return to Figure 5e-h. This\norganization is confusing and should be reconsidered for better coherence.\nReply and corresponding changes:\nThank you very much for your reminder! We have revised the manuscript to\nreorganize the figure sequence and corresponding text for improved logical flow and\nclarity.\nComment 6:\nThe authors should provide more information on how long it takes for BioGlue\nto form a pulmonary embolism in vivo.\nReply and corresponding changes:\nThanks for your valuable suggestions! In clinical practice, once BioGlue\ndetaches at the vascular adhesion site, the detached emboli would enter the\nbloodstream, quickly obstruct branching blood vessels and cause insufficient blood\nsupply to organs. To simulate the clinical embolus detachment and vascular embolism,\nwe established a rat pulmonary embolism model by injecting emboli via the posterior\nvena cava puncture. The emboli were gelled in vitro and punctured into the posterior\nvena cava of rats. Once entering the bloodstream, the emboli would flow with blood\ninto pulmonary circulation and rapidly occlude the pulmonary artery within seconds\nor minutes. This information has been added to the revised manuscript.\nRevised text\n…Once entering the bloodstream, the emboli would flow with blood into\npulmonary circulation and rapidly occlude the pulmonary artery within seconds or\nminutes... (Page 9)\nComment 7:\nThe rationale for using H9c2 cells, a cardiomyocyte cell line, for the biosafety\nevaluation should be explained, as this cell line may not be the most appropriate\nmodel for assessing BioGlue’s general biocompatibility.\nReply and corresponding changes:\nThanks for your valuable suggestions! In clinic practice, BioGlue is prevalently\nemployed in various cardiac procedures, such as proximal aortic dissection repair,\naortic root and arch reconstruction, valve repair and replacement surgery. H9c2 cells\nare one of the most used cell lines in cardiovascular system research, which were\nchosen for biosafety evaluation in this study. Meanwhile, as the reviewers suggest,\nanother commonly used cell line in cardiovascular research, human umbilical vein\nendothelia line (HUVEC cells), was employed in this study to evaluate the safety of\nthe Bi- BioGlue. The cell viabilities of H9c2 cells and HUVEC cells were remained\nhigher than 80% even at a concentration as high as 20 mg/mL Bi-BioGlue for 48 h,\nwhich results suggested low cytotoxicity of Bi-BioGlue towards both kinds of cells\n(Fig. 6b).\nRevised text\n…The in vitro cytotoxicity was investigated by standard MTT assay. H9c2 cells\nand HUVEC cells, two of the most commonly used cell lines in cardiovascular\nresearch, were cultured in media containing various concentrations of Bi-BioGlue for\n24 and 48 h. The cell viabilities of H9c2 cell and HUVEC cells lines remained higher\nthan 80% even at a concentration as high as 20 mg/mL Bi-BioGlue which results\nsuggested low cytotoxicity of Bi-BioGlue towards H9c2 cells and HUVEC cells (Fig.\nS16a, S16b). (Page 12)\nRevised figure\nFig. S16. Biosafety evaluation of Bi-BioGlue. Cell viabilities of H9c2 cells (a) and\nHUVEC cells (b) after incubated with different concentrations of Bi-BioGlue for 24\nor 48 h. (c) Body weight changes of control group and Bi-BioGlue group in 45 days.\nBio-chemical analysis (d) and routine blood tests (e) of control group, and\nBi-BioGlue group in 45 days. (f) H&E staining images of major organs (heart, liver,\nspleen, lung, and kidney) collected from the rats in control group and Bi-BioGlue\ngroup in 45 days.\nResponse to Reviewer 2\nComments:\nThe manuscript describes the characterization of a bismuth labelled derivative of\na clinically used surgical adhesive, and preclinical in vivo assessment of the\nfeasibility to visualize vascular emboli using this compound using CT imaging. The\nauthors show that the bismuth labelled derivative has the same adhesive capabilities\ncompared to the FDA approved BioGlue.\nComment 1:\nIn the introduction the size of emboli when damage occurs needs to be\nmentioned and related to the size detected in the results. The authors also need to\nexplain the benefit of hypersensitive detection of millimetre sized emboli. How do the\nauthors envision clinical application? Through watch and wait or intervention therapy?\nThe extent of symptoms will be dependent on the size of the embolism. Will there be\ndifferences in treatment compared to standard embolisms? As anticoagulants will not\nwork and nor will fibrinolytic therapy.\nReply and corresponding changes:\nThank you very much! The clinical impact of emboli is closely related to their\nsize. Large emboli (>5.0 mm) can cause life-threatening events such as pulmonary\nembolism or major stroke. Medium-sized emboli (1.0-5.0 mm) may lodge in smaller\nvessels of the lungs, brain, kidneys or retina, leading to localized ischemia and organ\ndysfunction. Even micro emboli around 1 mm, though initially asymptomatic, may\ntrigger chronic inflammation and cumulative damage, especially in sensitive organs.\nSince emboli of various sizes can pose significant safety risks, it is essential to detect\nemboli across a wide size range. Therefore, there is an urgent need for highly\nsensitive detection methods, particularly those capable of identifying micro emboli as\nsmall as 1 mm in diameter.\nTimely detection and early intervention of emboli significantly improve patient\noutcomes. During the use of surgical adhesives such as BioGlue, the intraoperative\nperiod represents the most critical window for embolus formation due to potential\ndetachment of adhesive fragments. Therefore, immediate post-operative imaging to\ndetect dislodged emboli is essential. Moreover, the early postoperative period also\ncarries a high risk for delayed embolus formation, highlighting the need for continued\nsurveillance. If translated into clinical practice, our proposed strategy would follow a\nclear detection timeline: a whole-body CT scan should be performed immediately\nafter surgery to screen for intraoperatively dislodged emboli, followed by regular CT\nscans during the early postoperative period to identify any subsequently formed\nemboli. This time-defined imaging protocol ensures that even ultra-small emboli can\nbe detected at high-risk stages, supporting timely clinical intervention.\nAnticoagulant and fibrinolytic therapy are important approaches for treating\nthrombosis, as they work by affecting platelets and related coagulation factors. In\nclinical practice, a range of therapeutic strategies are available for different types of\nemboli, including pharmacological thrombolysis, anticoagulant therapy, interventional\nthrombectomy, and surgical embolectomy. Thrombolytic therapy is commonly used\nfor acute thrombotic events (such as acute ischemic stroke or pulmonary embolism),\nfunctioning by activating the body's fibrinolytic system to dissolve the thrombus.\nAnticoagulant therapy is widely employed in the prevention and management of\nthromboembolic disorders such as venous thromboembolism and atrial\nfibrillation-related thrombi, primarily by inhibiting the coagulation cascade and\npreventing thrombus propagation. Interventional thrombectomy is typically applied in\ncases of acute large-vessel occlusion or when pharmacological therapies prove\ninsufficient, enabling rapid restoration of blood flow. Surgical embolectomy is\ngenerally reserved for massive or life-threatening emboli, such as severe pulmonary\nor peripheral arterial embolism.\nHowever, BioGlue is primarily composed of glutaraldehyde and cross-linked\nproteins, resulting in emboli with highly stable and degradation-resistant structures\nthat are unresponsive to conventional anticoagulant or thrombolytic therapies. At\npresent, interventional thrombectomy or surgical embolectomy are the only effective\nclinical options for removing BioGlue-derived emboli. Failure to detect and treat such\nemboli in a timely manner may lead to serious health consequences or life-threatening\ncomplications. Therefore, early detection and prompt intervention for BioGlue\nembolism are of critical importance in clinical management. This information has\nbeen added to the revised manuscript.\nRevised text\nAdhesive-derived emboli with different sizes (ranging from 1.0 mm to several\nmillimeters) … Besides, adhesive-derived emboli cannot be effectively treated by\nconventional anticoagulant or thrombolytic therapies and typically require\ninterventional or surgical removal. (Page 2)\nComment 2:\nA- Line 48: Optical imaging is not a suitable imaging methodology for this\napplication due to its limited penetration depth and should be excluded here.\nReply and corresponding changes:\nThank you very much for your suggestions! We have removed the description of\noptical imaging from the introduction in the revised manuscript.\nComment 3:\n- Line 53: The authors present the bismuth labelling as novel, but this has already\nbeen applied for adhesives. Tian et al (Advanced materials 2023) describe a\nbismuth-pectin organic-inorganic complex that was engineered into a transformable\nmicrogel (that they call bi-glue) and that is compatible with x-ray imaging. The\nstatement made by the authors needs to be revised and the paper by Tian et al should\nbe accurately referenced and the differences in approaches should be discussed.\nReply and corresponding changes:\nThank you very much! Tian et al. reported a bismuth-pectin organic-inorganic\ncomplex as an innovative contrast agent for gastrointestinal X-ray and CT imaging.\nTheir interesting work addresses a key limitation of conventional X-ray CT contrast\nagents, which lack adhesion to gastrointestinal tissues and are rapidly cleared after\noral administration, preventing complete imaging of the gastrointestinal tract. The\nbismuth-pectin complex developed by Tian et al. exhibits certain gastrointestinal\nadhesive properties, facilitating comprehensive X-ray and CT imaging throughout the\ndigestive tract.\nAlthough the material is named \"bi-glue\", the transformable microgel reported in\nthe referenced study is fundamentally a hydrogel system, not a surgical adhesive in\nthe conventional or clinical sense. The adhesion of the bismuth-pectin complex\nmerely slows the transit of the material through the gastrointestinal tract without\nphysically bonding different tissues together. Moreover, if such contrast agents were\nto possess the ability to bond disparate tissues like BioGlue, they would likely cause\nsevere adverse effects such as gastrointestinal adhesions, which are clinically\nunacceptable.\nIn contrast, the Bi-BioGlue we propose is a true surgical adhesive capable of\nfirmly bonding different tissues and is specifically designed for detecting emboli\ncaused by BioGlue detachment. Therefore, these two materials differ fundamentally\nin their research focus, scientific problems addressed, material properties, and\napplication scenarios. We have accurately cited this study and used it as a\nrepresentative example to describe its application area.\nRevised text\nIncorporating imaging units into adhesives or gels is a commonly employed\nstrategy for visualizing them through various imaging techniques like computed\ntomography (CT) imaging16-19, magnetic resonance imaging20, nuclear imaging17, 21,\nultra-sound imaging22-24, and photoacoustic imaging25-27. This approach has been\nextensively utilized to detect the location, morphology, and degradation behavior of\nadhesives and gels, and has found widespread application in tissue engineering,\ndisease diagnosis, drug delivery, and immunotherapy. (Page 3)\nComment 4:\nFig 3 shows the visualization of emboli over the course of 42 days. What was the\npercentage of resorbance of emboli? If all were resorbed, what is then the additional\nvalue of early detection?\nReply and corresponding changes:\nThank you very much! BioGlue is one of the most widely used surgical\nadhesives and is gradually resorbed in vivo over a period of months to even years.\nTherefore, Bi-BioGlue fixed at the wound site is inherently safe and will eventually\nbe metabolized by the body (J. Thorac. Cardiovasc. Surg., 2019, 157, 176). Figure 3\nshows Bi-BioGlue fixed at the wound site after surgical adhesion, rather than\nrepresenting detached emboli. Our 42-day monitoring study also demonstrated its\ngradual degradation and resorption over time. However, the situation is entirely\ndifferent for emboli. Once an embolus forms, it can immediately cause tissue damage\nor even life-threatening complications. As such, relying on the eventual resorption of\nadhesive-derived emboli is not a viable strategy. Instead, it is imperative to detect\nthese emboli at an early stage and promptly remove them through interventional or\nsurgical procedures.\nComment 5:\nAn explanation should be provided to how detection of millimeter sized emboli\nwill significantly improve the biosafety of surgical adhesions. In my opinion these\ntwo features are not directly linked as the biosafety profile of the adhesive is\ndependent on the chemical composition itself and not on its detectability. Figure 6\nshows biosafety assessment of Bi-BioGlue without comparison to the original\nBioGlue, therefore significant improvement cannot be claimed.\nReply and corresponding changes:\nThe biosafety of a surgical adhesive is not solely determined by its chemical\ncomposition; its potential to form undetectable emboli also poses a significant safety\nrisk. Therefore, the overall biosafety of an adhesive depends both on the\nbiocompatibility of its constituents and on the ability to detect and intervene in\nembolic events in a timely manner.\nBioGlue is primarily composed of bovine serum albumin and glutaraldehyde,\nand the albumin is inherently biocompatible. Although glutaraldehyde has known\nchemical toxicity, it forms covalent bonds with proteins and tissues during application,\nbecoming immobilized within the adhesive matrix, and is then slowly metabolized\nover time. As a result, its intrinsic chemical toxicity exerts minimal biological impact,\nwhich contributes to the high chemical safety profile of BioGlue and supports its FDA\napproval and widespread clinical use.\nHowever, the main biosafety concern lies in the potential formation of emboli\ncaused by dislodged BioGlue fragments, which are difficult to detect with\nconventional methods. This embolic risk represents a recognized clinical challenge in\nthe use of BioGlue. Therefore, in this study, we address this issue by incorporating\nBiOCl into BioGlue, enabling the real-time detection of even sub-millimeter emboli\nvia CT imaging. This strategy significantly reduces the potential risks associated with\nembolism and enhances the overall safety profile of BioGlue.\nIn terms of chemical composition, our development of Bi-BioGlue does not alter\nthe intrinsic biosafety profile of the original BioGlue. However, from the perspective\nof mitigating the risk of adverse events caused by dislodged emboli, Bi-BioGlue\noffers a significant safety advantage. While conventional BioGlue lacks the ability to\nvisualize emboli once detached, Bi-BioGlue enables timely detection of emboli\nthrough CT imaging, allowing for prompt clinical intervention. Therefore, our\napproach improves the overall safety of BioGlue not by changing its chemical\ncomposition but by solving one of its most critical clinical challenges, which is the\ninability to detect embolic complications. By enabling reliable post-operative\nmonitoring of emboli, Bi-BioGlue provides a more comprehensive and proactive\nsafety profile. Thus, our approach provides a scientifically grounded and rational\nsolution to improve the biosafety of BioGlue through the integration of\nembolus-traceable contrast functionality.\nRegarding Figure 6, our primary objective was to evaluate whether the\nincorporation of BiOCl introduces any conventional biosafety concerns, including\neffects on body weight, serum biochemistry, hematology, and histopathology. The\ncomprehensive toxicological evaluation provided strong evidence supporting the\nexcellent biocompatibility of BiOCl. However, we would like to emphasize that\nconventional biosafety assessment is not the main focus of our study. This work aims\nto solve the critical clinical challenge of detecting adhesive emboli caused by BioGlue\ndetachment, a risk that is currently difficult to manage. The significant improvement\nin biosafety we claim is not based on standard toxicity profiles, but rather on the\nability of Bi-BioGlue to enable highly sensitive and timely detection of emboli,\nwhich directly contributes to reducing embolism-related risks.\nThis information has been added to the revised manuscript.\nRevised text\n…Therefore, our approach enhances BioGlue's safety by addressing the clinical\nchallenge of undetectable embolic complications, rather than altering its chemical\ncomposition. By enabling reliable post-operative monitoring of emboli, Bi-BioGlue\nprovides a more comprehensive and proactive safety profile. (Page 10)\n…The systematic toxicological assessment demonstrated Bi-BioGlue does not alter\nthe intrinsic biosafety profile of the original BioGlue in terms of chemical\ncomposition. (Page 13)\nComment 6:\nStability assessment of the created complex is insufficient. How can the authors\nensure that the complex will remain stable in vivo? This should at least be tested\nunder comparable conditions.\nReply and corresponding changes:\nThank you very much! We systematically studied the morphological changes of\nBi-BioGlue after incubation in various simulated physiological media over different\ntime periods, and assessed the release of Bi ions. The experimental results\ndemonstrated that Bi-BioGlue maintained a stable morphology throughout the\nmonitoring period with negligible Bi3+ release. These findings indicate that BiOCl\nexhibits efficient labeling within the BioGlue matrix, and the resulting Bi-BioGlue\npossesses excellent physicochemical stability.\nIn vivo experiments further revealed a gradual decline in the CT signal of\nBi-BioGlue over time, indicating a slow degradation process in physiological\nenvironments. This slow degradation is primarily attributed to the continuous\nmovement of body fluids, which promotes material transport and diffusion-consistent\nwith the intrinsic long-term degradation behavior of BioGlue itself. Notably, this\ngradual degradation provides a sufficient imaging window for the detection of emboli\nboth intraoperatively and during the early postoperative high-risk period.\nIn contrast, other modified adhesives such as Bi-DTPA-BioGlue and I-BioGlue\nexhibit rapid release of contrast agents, resulting in a sharp decline in CT signal\nintensity, thereby failing to support stable embolus imaging and timely detection. In\nsummary, we have provided a more accurate description of the stability of Bi-BioGlue:\nit exhibits high labeling efficiency and adequate in vivo stability, enabling a reliable\nimaging time window for effective detection of emboli.\nRevised text\n…In addition, the morphological changes of Bi-BioGlue after incubation in\nvarious simulated physiological media over different time periods and the release of\nBi ions were systematically studied. The experimental results demonstrated that\nBi-BioGlue maintained a stable morphology throughout the monitoring period, with\nnegligible Bi3+ release (Fig. S5, S6). …These findings indicate that BiOCl exhibits\nefficient labeling within the BioGlue matrix, and the resulting Bi-BioGlue possesses\nexcellent physicochemical stability. (Page 6)\nRevised figure\nFig. S6. The release curves of Bi elements in Bi-BioGlue within 14 days across\ndifferent media including H O, PBS, H O , and serum.\n2 2 2\nComment 7:\nHow is the apparent degradation of the adhesive being taken into account? It\nneeds to be investigated that the Bi remains complexed during degradation. The\npresence of non-labelled adhesive and a possible effect on emboli formation and\nvisualization needs to be assessed.\nReply and corresponding changes:\nThank you very much for your constructive comments. BiOCl was selected as a\nhigh-performance imaging label due to its favorable physicochemical properties,\nenabling uniform labeling of BioGlue. In vitro studies, including CT imaging and\nelemental mapping, confirmed the homogeneous distribution of BiOCl within the\nBioGlue matrix, with no evidence of unlabeled regions. Stability assessments further\ndemonstrated that BiOCl was not rapidly released from Bi-BioGlue, indicating strong\nretention of the BiOCl component. During in vivo application, the two primary\ncomponents, BiOCl-BSA mixture and glutaraldehyde, rapidly reacted upon contact,\nforming a solidified Bi-BioGlue within seconds. Although BioGlue inherently\nundergoes slow degradation in vivo, in vivo CT imaging revealed that Bi-BioGlue\nmaintained stable morphology and size within the first week, with only a gradual\ndecrease in CT signal intensity. This indicates that the initial degradation of\nBi-BioGlue is very slow and that there is no apparent presence of unlabeled adhesive.\nAs time progressed, Bi-BioGlue exhibited further degradation, as evidenced by a\ngradual reduction in size on CT imaging. Given the negligible Bi3+ release of BiOCl\nunder simulated physiological conditions in vitro, it is reasonable to infer that BiOCl\nretains considerable labeling efficiency even during later stages of degradation.\nConsidering that the intraoperative and early postoperative periods represent\nhigh-risk windows for embolus detachment when using BioGlue, the demonstrated in\nvivo labeling efficiency of Bi-BioGlue during the early stage ensures the reliable\ndetection of emboli within this critical timeframe.\nRevised text\n…The Bi-BioGlue maintained stable morphology and size within the first week,\nwith only a gradual decrease in CT signal inten-sity. This indicates that the initial\ndegradation of Bi-BioGlue is very slow and that there is no apparent presence of\nunlabeled adhesive. As time progressed, Bi-BioGlue exhibited further degradation, as\nevidenced by a gradual reduction in size on CT imaging, eventually the CT value of\nadhesion site…Given the negligible Bi3+ release of BiOCl under simulated\nphysiological conditions in vitro, it is reasonable to infer that BiOCl retains\nconsiderable labeling efficiency even during later stages of degradation (Fig. S11).\nConsidering that the intraoperative and early postoperative periods represent high-risk\nwindows for embolus detachment when using BioGlue, the demonstrated in vivo\nlabeling efficiency of Bi-BioGlue during the early stage ensures the reliable detection\nof emboli within this critical timeframe. (Page 8)\nRevised figure\nFig. S11. The release curves of Bi elements in BiOCl within 14 days across different\ndispersion systems including H O, PBS, H O , and serum.\n2 2 2\nResponse to Reviewer 3\nThe authors present a bismuth-based BiOCl-doped BioGlue for detecting\nvascular emboli originating from surgical adhesives using CT imaging. This\nCT-visible adhesive enables in vivo tracking of adhesive degradation and the\nreal-time detection of small emboli. The Bi-BioGlue demonstrates enhanced stability,\nbiocompatibility, and prolonged imaging persistence, with the capability to detect\nemboli as small as 1.2 mm in a pulmonary embolism model. Overall, this study is\nwell-designed with a rigorous methodology. However, several questions and\nsuggestions need to be addressed before considering publication.\nReply:\nThank you very much for your such positive comments!\nComment 1:\nThe authors used known molecules to form a crosslinked glue. However, the\nchemistry should be shown more clearly, including the chemical structures of each\nmolecule and the detailed characterizations after synthesis.\nReply and corresponding changes:\nThank you very much! Based on the reviewer's suggestions, we have presented\nthe chemical structure of each molecule in the figure. Additionally, BioGlue is\ncomposed of purified bovine serum albumin (45%, w/v) and glutaraldehyde (10%,\nw/v), which are mixed in a dual-barrel syringe and applied to the surgical site. The\nprinciple behind BioGlue is that glutaraldehyde forms a stable seal by covalently\nlinking to the lysine amino acid fragment in bovine serum albumin, and it can also\nconnect to lysine residues in other proteins in the tissue, resulting in excellent tissue\nadhesion (Figure 2a).\nRevised text\n…BioGlue is composed of purified bovine serum albumin (45%, w/v) and\nglutaraldehyde (10%, w/v), which are mixed in a dual-barrel syringe and applied to\nthe surgical site. The principle behind BioGlue is that glutaraldehyde forms a stable\nseal by covalently linking to the lysine amino acid fragment in bovine serum albumin,\nand it can also connect to lysine residues in other proteins in the tissue, resulting in\nexcellent tissue adhesion. (Page 5)\nRevised figure\nFig. 2. (a) Synthetic scheme of the Bi-doping BioGlue through the GA-induced\ncross-linking method.\nComment 2:\nThe cross-linking mechanism between BiOCl and the BioGlue matrix is briefly\nmentioned, but more experimental details are required. This should include the\nkinetics and degree of cross-linking, as these factors could directly influence adhesion\nstrength and stability. Additionally, as BiOCl stability is crucial for sustained imaging,\nplease discuss the stability of BiOCl in the adhesive under physiological pH and\noxidative conditions.\nReply and corresponding changes:\nThank you very much! Bi-BioGlue and BioGlue share the same crosslinking\nmechanism, which is based on the covalent interaction between BSA and GA. The\nonly difference lies in the composition of the protein component: Bi-BioGlue\nincorporates BSA doped with BiOCl, whereas BioGlue utilizes native BSA. Notably,\nBiOCl is physically encapsulated within the BioGlue matrix and does not participate\nin the chemical crosslinking process.\nAs suggested by the reviewers, we systematically evaluated the crosslinking\nkinetics of both BioGlue and Bi-BioGlue by rheological analysis. The results\ndemonstrated that upon mixing, the BSA and glutaraldehyde components exhibited\nsimilarly rapid crosslinking kinetics, forming an opaque white solid within seconds.\nFurthermore, tissue adhesion tests showed that Bi-BioGlue exhibited comparable\nadhesive strength to BioGlue. These findings indicate that the incorporation of BiOCl\ndoes not interfere with the crosslinking behavior or adhesive performance of BioGlue.\nAs suggested by the reviewer, we evaluated the structural stability of Bi-BioGlue\nunder different simulated physiological conditions. Specifically, both Bi-BioGlue and\nBiOCl were incubated in four representative media: deionized water (H O),\n2\nphysiological buffer (10 mM PBS, pH 7.4), an oxidative stress environment (1 mM\nH O ), and serum. At predetermined time points, we performed X-ray diffraction\n2 2\n(XRD) analysis and quantified the release of Bi3+ ions to assess structural integrity\nand potential degradation. The results showed that BiOCl, whether incorporated into\nBi-BioGlue or not, retained its crystalline structure across all media without\nnoticeable changes. Moreover, the release of Bi3+ ions remained below 2% in all\nconditions. Additionally, the morphology and dimensions of Bi-BioGlue exhibited no\nobvious alterations during the incubation period. Collectively, these findings\ndemonstrate the excellent structural and chemical stability of both BiOCl and\nBi-BioGlue under physiologically relevant environments.\nRevised text and figures\n…Bi-based BioGlue and BioGlue share the same crosslinking mechanism, which\nis based on the covalent interaction between BSA and GA. The only difference lies in\nthe composition of the protein component: Bi-based BioGlue incorporates BSA doped\nwith Bi-based imaging probes, whereas BioGlue utilizes native BSA… (Page 5)\nThe crosslinking kinetics of both BioGlue and Bi-BioGlue by rheological\nanalysis demonstrated that upon mixing, the BSA and glutaraldehyde components\nexhibited similarly rapid crosslinking kinetics, forming an opaque white solid within\nseconds (Fig. 2h). Further XRD analysis showed that BiOCl, whether incorporated\ninto Bi-BioGlue or not, retained its crystalline structure across all media without\nnoticeable changes (Fig. S7). (Page 6)\nFig. S7. XRD patterns of Bi-BioGlue after incubation in different media: H O(a),\n2 2\nincubation in different dispersion systems: H O (e), PBS (f), H O (g), and serum (h)\n2 2 2\non days 1, 7, and 14.\nComment 3:\nOn page 6, line 105, please include a reference for the synthesis method of\nBi-DTPA.\nReply and corresponding changes:\nThank you very much! The reference for the synthesis of Bi-DTPA has been\ncited in the revised manuscript.\nComment 4:\nIn Fig S2, please add or label BioGlue, gelatin, and sodium alginate within the\nfigures.\nReply and corresponding changes:\nThank you very much for your suggestion! BioGlue, gelatin, and alginate have\nbeen marked in the figures of the revised supporting information.\nRevised figure\nFig. S2. Photographs of BiOCl-doped BioGlue, Chitosan-gelatin and sodium\nalginate-Ca2+ with different concentrations (0-88 mM) of BiOCl.\nComment 5:\nIn Fig. 2g, please clarify what the colors represent.\nReply and corresponding changes:\nThank you very much for your advice! We clarified the element corresponding to\neach color. In Fig. 2c, orange represents Cl element, yellow represents Bi element,\nand purple represents S element. The element mapping image clearly shows the\ndistribution of Cl, Bi, and S elements in Bi-BioGlue.\nRevised text\nElemental mapping images demonstrated the presence and uniform distribution\nof Cl (orange), Bi (yellow), and S (purple) in the freeze-dried Bi-BioGlue (Fig. 2g).\n(Page 6)\nComment 6:\nIn Fig. 2k, what are the concentrations of Bi-BioGlue and iohexol-doped\nBioGlue used in this study? If the concentration is 105 mM, please explain the\nrationale for this choice. Additionally, please provide a more detailed comparison of\ndetection sensitivity with standard iodine-based agents.\nReply and corresponding changes:\nThank you very much! We systematically optimized the Bi doping concentration\nto assess its effect on the CT imaging performance of Bi-BioGlue. Increasing the Bi\ncontent enhanced the sensitivity of CT detection; however, excessive doping led to\nundesirable changes in the colloidal properties and raised potential concerns about\nbiosafety. Our results showed that the CT value of bulk Bi-BioGlue gradually\nincreased with rising Bi concentrations. When the Bi concentration reached 105 mM,\nthe CT value of Bi-BioGlue reached approximately 659 HU, yielding a favorable\nsignal-to-background ratio compared to normal tissues, which typically exhibit CT\nvalues in the range of 40-80 HU. It should be noted that maintaining a high CT value\nin bulk Bi-BioGlue relative to soft tissue is essential, as the formation of very small\nBioGlue emboli will lead to a certain degree of CT signal attenuation due to partial\nvolume effects. Importantly, at this concentration, the mechanical and adhesive\nproperties of BioGlue were not adversely affected. Therefore, 105 mM was selected\nas the optimal Bi doping concentration for the following studies.\nIndeed, we had provided a detailed comparison of detection sensitivity with\nstandard iodine-based agents in original manuscript. On conventional CT, though CT\nintensity of both Bi-BioGlue and I-BioGlue was linear enhancement with the\nincreasement of concentration, the Bi-BioGlue yielded a higher CT intensity\ncompared to I-BioGlue on same Bi/I-doped concentration at commonly used tube\nvoltage in clinic (120 kV) (Fig. 2e and Fig. S8). During spectral CT scanning, Due to\nthe much higher K edge energy value of Bi (91 keV) compared to the I (33.2 keV),\nthe CT intensity of I-BioGlue showed sharply attenuation decay curve and at high\nmonochromatic X-ray energy on spectral CT imaging, while the Bi-BioGlue exhibited\na relatively flat decay curve. At 190 KeV, the CT values of Bi-BioGlue was 3.9 times\nhigher than that of iohexol-doped BioGlue. In terms of doping stability, since clinical\niodine-based contrast agents are small molecules with excellent water solubility,\nI-BioGlue exhibited a burst release behavior of imaging probes similar to that of\nBi-DTPA-BioGlue when immersed in water. These results indicate that clinical\niodine-based contrast agents are unsuitable for labeling surgical adhesives due to their\nrelatively poor sensitivity and labeling capability.\nRevised text\nBi doping concentration was systematically optimized to assess its effect on the\nCT imaging performance of Bi-BioGlue. Increasing the Bi content enhanced the\nsensitivity of CT detection; however, excessive doping led to undesirable changes in\nthe colloidal properties and raised potential concerns about biosafety. The result\nshowed that the CT value of bulk Bi-BioGlue gradually increased with rising Bi\nconcentrations. When the Bi concentration reached 105 mM, the CT value of\nBi-BioGlue reached approximately 659 HU, yielding a favorable\nsignal-to-background ratio compared to normal tissues, which typically exhibit CT\nvalues in the range of 50-80 HU. It should be noted that maintaining a high CT value\nin bulk Bi-BioGlue relative to soft tissue is essential, as the formation of very small\nBioGlue emboli will lead to a certain degree of CT signal attenuation due to partial\nvolume effects. Therefore, 105 mM was selected as the optimal Bi doping\nconcentration for the following studies. (Page 5)\nComment 7:\nIn Fig. S3, the color change in DMEM from D0 to D14 (red to yellow) likely\nreflects pH changes. Please provide an explanation of any chemical reactions or\ndegradation processes that may have occurred during the time period.\nReply and corresponding changes:\nThank you very much! In accordance with the reviewer's suggestions, we\nrepeated the DMEM static stability experiment under more stringent and rigorous\nconditions, and included a BioGlue group for comparison. The color of DMEM\nincubated with Bi-BioGlue gradually lightened over 14 days, without showing any\nsigns of yellowing. A similar color change was also observed in DMEM incubated\nwith BioGlue. It is worth noting that in our previous experiments, yellowing of\nDMEM was observed when incubated with Bi-BioGlue over the same period, which\nwas most likely caused by bacterial contamination. These findings indicate that the\ncolor change of DMEM is unrelated to the presence of BiOCl. In addition, we\nmeasured the pH of the solutions and found no significant changes. Given that phenol\nred, the pH indicator in DMEM, does not chemically react with any components of\nBi-BioGlue, we infer that the lightening of DMEM color is primarily caused by the\nphysical adsorption of phenol red by Bi-BioGlue.\nRevised figure\nFig. S5. Photographs of Bi-BioGlue immersed in PBS, normal saline and DMEM for\n14 days. Photographs BioGlue immersed in DMEM for 14 days.\nComment 8:\nOn page 7, line 141, verify if Fig. 1c is correctly referenced.\nReply and corresponding changes:\nThank you very much for pointing out the error! We apologize for the mistake\nmade here and have corrected the erroneously cited Fig. 1c to Fig. 2e.\nComment 9:\nIn Fig. S6, please provide experimental details, including bonding duration and\nthe time elapsed before testing Bi-BioGlue.\nReply and corresponding changes:\nThank you very much! To evaluate the tissue adhesive ability of Bi-BioGlue,\nwe investigated its adhesive properties on pig aorta vessels ex vivo. Bi-BioGlue was\ninjected into the leakage site using a syringe, with an adhesion time of 30 s. After the\nadhesion, the system was allowed to stabilize for 120 s before red ink, simulating\nblood, was injected from the upper end. No \"blood\" was observed to leak, thereby\nconfirming the excellent adhesive capability of Bi-BioGlue.\nRevised text\nEx Vivo Tissue Adhesion of Bi-BioGlue. To evaluate the tissue adhesive\nperformance of Bi-BioGlue, an ex vivo leakage model was established using isolated\npig aorta vessels. A longitudinal incision was made on the vessel wall to simulate a\nleakage site. Bi-BioGlue was applied to the defect using a syringe, followed by 30 s\nof contact to initiate adhesion. The repaired vessel was then allowed to stand\nundisturbed for 120 s to ensure complete gelation and bonding. Subsequently, red ink\nwas injected from the proximal end of the vessel to simulate blood flow. The absence\nof ink leakage from the repair site was used as an indicator of effective tissue\nadhesion. (Page 14)\nComment 10:\nIn Fig. 3f, indicate the time required to achieve sealing and hemostatic effects.\nThis information should be included in the experimental section or figure legend.\nReply and corresponding changes:\nThank you for your attention to this issue. Both BioGlue and Bi-BioGlue can\nseal and stop liver bleeding within about 30 s, and the time of hemostasis has been\nmarked in the experimental section.\nRevised text\n…Both adhesives sealed the bleeding site and stopped hemorrhage within\napproximately 30 s… (Page 15)\nComment 11:\nIn Fig. 4a, provide more details regarding the preparation of Bi-BioGlue emboli\nin air and in red meat.\nReply and corresponding changes:\nThank you very much! We conducted an investigation into the limits of CT\nimaging detection of thrombi in air and red meat. First, the gel was allowed to\npolymerize in a petri dish, after which it was carefully trimmed into cubic gel blocks\nof varying sizes. Then, we placed the emboli either in air or sandwiched between two\nslices of red meat with defined thickness for CT scanning.\nRevised text\nBi-BioGlue was carefully trimmed into cubic emboli with diameters of 1.0, 1.2,\n2.0, 3.0, 4.0, and 5.0 mm. CT imaging was conducted under various conditions to\nevaluate the detectability of emboli in ex vivo settings. (Page 15)\nDetection limits in air and tissue: Emboli were placed either in air or sandwiched\nbetween two 2 cm-thick slices of red meat. Scanning was performed with a FOV of\n256 × 256 mm, a slice thickness of 0.5 mm, and adaptive tube current at 120 kV.\n(Page 16)\nComment 12:\nIn Fig. 5a, describe the process for creating Ca (PO ) -doped BioGlue.\n3 4 2\nReply and corresponding changes:\nThank you very much! The Ca (PO ) -doped BioGlue is prepared as follows.\n3 4 2\nFirst, 4.5 g of Ca (PO ) and 4 g of BSA are weighed and dissolved in water to form\n3 4 2\nCa (PO ) /BSA mixture (final volume 10 mL, Ca (PO ) 1.45 mol/L, BSA 400\n3 4 2 3 4 2\nmg/mL), followed by stirring evenly. It is worth noting that during the preparation of\ncalcium phosphate-doped BioGlue, the concentration of BSA used was 400 mg/mL\nrather than 450 mg/mL. This adjustment was made because a higher BSA\nconcentration, combined with calcium phosphate, is difficult to uniformly disperse in\nwater. Since the CT contrast primarily arises from the calcium content, the slight\nreduction in BSA concentration is not expected to affect the experimental outcomes.\nNext, Ca (PO ) /BSA -doped BioGlue was formed by reacting a mixture of\n3 4 2\nCa (PO ) /BSA and 10% glutaraldehyde in a 96-well plate, with volumes of 160\n3 4 2\nμL/well and 40 μL/well, respectively.\nRevised text\n…4.5 g of Ca (PO ) and 4 g of BSA are weighed and dissolved in water to form\n3 4 2\nCa (PO ) /BSA mixture (final volume 10 mL, Ca (PO ) 1.45 mol/L, BSA 400\n3 4 2 3 4 2\nmg/mL), followed by stirring evenly. It is worth noting that during the preparation of\nCa (PO ) -doped BioGlue, the concentration of BSA used was 400 mg/mL rather than\n3 4 2\n450 mg/mL. This adjustment was made because a higher BSA concentration,\ncombined with calcium phosphate, is difficult to uniformly disperse in water. Since\nthe CT contrast primarily arises from the calcium content, the slight reduction in BSA\nconcentration is not expected to affect the experimental outcomes. Next,\nCa (PO ) /BSA -doped BioGlue was formed by reacting a mixture of Ca (PO ) /BSA\n3 4 2 3 4 2\nand 10% glutaraldehyde in a 96-well plate, with volumes of 160 μL/well and 40\nμL/well, respectively… (Page 16)\nComment 13:\nOn page 15, line 317, ensure consistency in units, as “1.2 mm” appears in several\nplaces. Please clarify if this measurement refers to diameter or volume.\nReply and corresponding changes:\nThank you very much! I apologize for the mistake in the text. We clarified that\nthe term \"1.2 mm\" throughout the manuscript refers to the dimension (size) rather\nthan the volume.\nRevised text\n…Ex vivo CT imaging showed that emboli with diameters of 1.0 mm (edge\nlength of cubic emboli) and 1.2 mm… (Page 8)\nComment 14:\nOn pages 9 and 12, where descriptions for Fig. 4 and Fig. 5 are located,\nreorganize the paragraphs to improve flow and readability.\nReply and corresponding changes:\nThank you very much for your reminder on this issue! We have revised the\nmanuscript to reorganize the figure sequence and corresponding text for improved\nlogical flow and clarity.\nOther corrections\nTo demonstrate the expandability of this study, the proposed synthesis strategy\nwas further extended to the preparation of other metal (Hf/Ta/Yb)-doped BioGlue\n(Fig. S3). Dynamic oscillation time sweep measurements showed that exhibit similar\nG'/G'' ratios, indicating that doping with metal elements does not affect the\nrheological properties of the adhesives obviously (Fig. S4). (Page 6)\nSynthesis of Hf-, Ta-, Yb-doped BioGlue. To synthesize Hf-, Ta-, and\nYb-doped BioGlue, HfCl , TaCl , and YbCl were individually mixed and stirred with\n4 5 3\nBSA solution to obtain metal probe/BSA mixtures containing 150 mM of the metal\nelement and 450 mg/mL of BSA, respectively. Glutaraldehyde was then added at a\nvolume ratio of 4:1 to form the corresponding Hf-, Ta-, and Yb-doped BioGlue. (Page\n14)\nRevised figures\nFig. S3. Photographs and corresponding CT images of conventional BioGlue,\nHf-BioGlue, Ta-BioGlue and Yb-BioGlue with the Hf, Ta, and Yb concentration of\n105 mM.\nFig. S4. Dynamic oscillatory time sweep measurements of BioGlue, Hf-BioGlue,\nTa-BioGlue and Yb-BioGlue."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors provided additional experimental results to address part of the comments provided by the reviewers. However, the additional data did not cover all issues raised and a number of comments (for all three reviewers) were not or not accurately addressed. Unfortunately, previous questions raised on the novelty of the approach and the degree of translational advance have not been resolved.\nThe authors are also encouraged to reply to the provided comments only and in a short and concise manner, and exclude lengthy text sections that are not incorporated in the manuscript.", "reviewer_3": "The authors have made substantial efforts to improve this manuscript, and I believe the revised version is now suitable for publication in its current form.", "reviewer_5": "This manuscript reported CT imaging-based hypersensitive detection of single millimeter vascular emboli from adhesive in vivo. The proposed Bi-BioGlue with good CT imaging capability can monitor the location, morphology, and degradation process of Bi-BioGlue by CT imaging for a long time, and pulmonary emboli caused by Bi-BioGlue, with size as small as 1.2 mm, can be successfully detected. Additionally, the Bi-Biogule enabled precise detection of pulmonary emboli in spectral CT imaging, unaffected by confounding calcifications. Overall, this study provides a promising non-invasive detection strategy for adhesive emboli.\nAfter the first round of revision, the reviewers’ comments were fully addressed, and the quality and level of the manuscript were significantly enhanced. I would like to recommend it for publication in Nature Communications after minor revisions.\nMinor issues:\n1. In Fig. 2f, the FTIR spectrum of BioGlue needs to be supplemented to further illustrate the impact of the introduction of BiClO on BioGlue.\n2. The color of the wells in the 96-well plate in Fig. 5a that contain I, Bi, and BG should use colors with greater distinction, corresponding to the colors in Fig. 5d, which would make it easier to understand.\n3. This study achieved good results in animals. However, if it is to be applied in clinical practice in the future, many challenges will still need to be overcome. I suggest that the authors summarize the limitations of this study and the future directions.\n4. There are some inconsistencies in formatting and grammatical errors, for instance, both “Bioglue” and “BioGlue” are used. The authors should carefully review the manuscript throughout and make necessary revisions.", "reviewer_6": "The authors incorporated Bi element in commercial surgical adhesives (BioGlue) for noninvasive detection of adhesive-induced emboli using spectral CT imaging. The results clearly demontrated that the detecting time is over than 40 days, and the size could limit to 1.2 mm. The authors have carefully revised all the comments. So, in my opinon, the revision should be published as it is now."}, "author_response": "Response to Reviewer’s Comments\nResponse to Reviewer 2\nComments:\nThe authors provided additional experimental results to address part of the comments provided by the reviewers. However, the additional data did not cover all issues raised and a number of comments (for all three reviewers) were not or not accurately addressed. Unfortunately, previous questions raised on the novelty of the approach and the degree of translational advance have not been resolved.\nThe authors are also encouraged to reply to the provided comments only and in a short and concise manner, and exclude lengthy text sections that are not incorporated in the manuscript.\nReply and corresponding changes:\nThank you very much for your careful evaluation and thoughtful insights. To address the reviewers’ concerns regarding innovation and clinical translational potential, we provide the following clarification and explanation. Regarding innovation, the innovation of this work lies in providing an early and accurate diagnostic solution for the severe complication of detached emboli caused by the clinically widely used adhesive BioGlue. Through systematic and comprehensive optimization, we identified amorphous BiOCl as a CT labeling probe that can stably label BioGlue without affecting its performance. Systematic in vivo results fully demonstrate that this method can achieve accurate detection of single millimeter detached emboli. Even under conditions that mimic the presence of pulmonary calcified nodules, advanced spectral CT imaging still enables accurate and interference free detection of detached emboli. This study provides an important solution for the safe use of BioGlue and is also the first report to achieve accurate detection of single millimeter scale detached emboli.\nRegarding clinical translational potential, modifying and improving biomaterials or drugs that have already been used clinically is a common strategy for achieving rapid clinical translation. This study introduces a BiOCl labeling probe with good biosafety on the basis of BioGlue, therefore demonstrating good clinical translational potential. In the future, we will further conduct in depth studies of the long-term biosafety of Bi BioGlue in large animals, explore its actual clinical translational potential and promote its clinical translation.\nResponse to Reviewer 3\nComments:\nThe authors have made substantial efforts to improve this manuscript, and I believe the revised version is now suitable for publication in its current form.\nReply and corresponding changes:\nThank you very much for your positive comments.\nResponse to Reviewer 5\nComment 1:\nIn Fig. 2f, the FTIR spectrum of BioGlue needs to be supplemented to further illustrate the impact of the introduction of BiClO on BioGlue.\nReply and corresponding changes:\nThank you for your valuable comment. We measured the FTIR spectrum of BioGlue, which is included in revised Fig. 2f.\nRevised figure in manuscript\nComment 2:\nThe color distinction in the wells of Fig. 5a should be clearer and correspond to the colors in Fig. 5d.\nReply and corresponding changes:\nThank you for your helpful comment. We have enhanced the color contrast in Fig. 5a so that the different groups are clearly distinguishable. The updated panel is now consistent with the color scheme used in Fig. 5d.\nRevised figure in manuscript\nComment 3:\nThe authors should summarize the limitations of the study and the future directions.\nReply and corresponding changes:\nThank you for your valuable comment. A concise summary of the limitations and future prospects has been added to the last paragraph of Results and Discussion section. Although this study provides a highly sensitive strategy for detecting BioGlue emboli, the current work remains limited to a rat model. In addition, the emboli used in this study were pre-fabricated rather than naturally formed during clinical BioGlue procedures. The biosafety evaluation of Bi BioGlue is also still relatively basic. In future work, we will perform BioGlue adhesion procedures in large animals such as pigs to simulate the natural formation of emboli and to assess the detection capability of this approach under clinically relevant conditions. We will also conduct more comprehensive long-term evaluations of the biosafety of Bi BioGlue to advance its potential for clinical translation.\nRevised text in manuscript：\n…Although this study provides a highly sensitive strategy for detecting BioGlue emboli, the current work remains limited to a rat model. In addition, the emboli used in this study were pre-fabricated rather than naturally formed during clinical BioGlue procedures. The biosafety evaluation of Bi BioGlue is also still relatively basic. In future work, we will perform BioGlue adhesion procedures in large animals such as pigs to simulate the natural formation of emboli and to assess the detection capability of this approach under clinically relevant conditions. We will also conduct more comprehensive long-term evaluations of the biosafety of Bi BioGlue to advance its potential for clinical translation.\nComment 4:\nThere are inconsistencies in formatting and grammatical errors, such as the use of both “Bioglue” and “BioGlue”.\nReply and corresponding changes:\nWe have carefully proofread the manuscript and corrected all terminology and spelling inconsistencies to ensure full uniformity throughout the text and figures.\nResponse to Reviewer 6\nComments:\nThe authors incorporated Bi element in commercial surgical adhesives (BioGlue) for noninvasive detection of adhesive-induced emboli using spectral CT imaging. The results clearly demonstrated that the detecting time is over than 40 days, and the size could limit to 1.2 mm. The authors have carefully revised all the comments. So, in my opinion, the revision should be published as it is now.\nReply and corresponding changes:\nThank for your such positive comments."}]

Decision letter