Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Cotton swabs wrapped with three-dimensional silver nanoflowers as SERS substrates for the determination of food colorant carmine on irregular surfaces.

A lightweight, portable, low-cost, and accessible cotton swab was employed as surface enhanced Raman spectroscopy (SERS) matrix template. The silver nanoflowers were in situ grown on the surface of cotton swabs to form three-dimensional Ag nanoflower@cotton swabs (AgNF@CS) SERS substrate with high-density and multi-level hot spots. The SERS performance of AgNFs@CS substrates with various reaction time was systematically studied. The optimal AgNF-120@CS SERS substrate exhibits superior detection sensitivity of 10-10 M for methylene blue, good signal reproducibility, high enhancement factor of 1.4 × 107, and excellent storage stability (over 30 days). Moreover, the AgNF-120@CS SERS substrate also exhibits prominent detection sensitivity of 10-8 M for food colorant of carmine. Besides, the portable AgNF-120@CS SERS substrate is also capable of detecting food colorant residues on irregular food surfaces.

Concurrence of novel mutations causing Gilbert's and Dubin-Johnson syndrome with poor clinical outcomes in a Han Chinese family.

Dual-hereditary jaundice (Dubin-Johnson syndrome (DJS) and Gilbert's syndrome (GS)) is a rare clinical entity resulting from defects of the ATP binding cassette subfamily C member 2 (ABCC2) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1) genes with autosomal recessive inheritance. In this study, we aimed to investigate the mutation profiles and characterize the phenotypes in a Han Chinese family with DJS and GS. Genetic screening for variants in the ABCC2 and UGT1A1, immunohistochemistry for expression of ABCC2, and histopathological examination were carried out. The proband and his brother had unconjugated and conjugated hyperbilirubinemia after birth. The proband's sister had only conjugated hyperbilirubinemia after birth. The proband developed into pleural effusions and ascites, pericardial thickening, intrahepatic and extrahepatic biliary duct dilatation, and enlarged gallbladder at age 50. Hepatocellular carcinoma occurred in the proband's brother at age 46. Seven compound defects of the ABCC2 gene [c.2414delG, p.(Ile1489Gly), p.(Thr1490Pro), and p.(Ile1491Gln)] and the UGT1A1 gene (c.-3279T>G, p.(Gly71Arg), and p.(Pro451Leu)) were identified in family members. Accumulation of pigment in hepatocytes characteristic of that in DJS was present in the proband and his brother. Expression of ABCC2 protein was markedly diminished in the patient's liver. Our results show a different genetic profile of DJS and GS in a Han Chinese family, indicating a more complex pattern of dual-hereditary jaundice among different populations. The present study illuminates the underpinnings of DJS and GS and extends the mutation profiles and phenotypes of these two syndromes in dual-hereditary jaundice.

Illuminating the path: aggregation-induced emission for food contaminants detection.

Food safety is a global concern that deeply affects human health. To ensure the profitability of the food industry and consumer safety, there is an urgent need to develop rapid, sensitive, accurate, and cost-effective detection methods for food contaminants. Recently, the Aggregation-Induced Emission (AIE) has been successfully used to detect food contaminants. AIEgens, fluorescent dyes that cause AIE, have several valuable properties including high quantum yields, photostability, and large Stokes shifts. This review provides a detailed introduction to the principles and advantages of AIE-triggered detection, followed by a focus on the past five years' applications of AIE in detecting various food contaminants including pesticides, veterinary drugs, mycotoxins, food additives, ions, pathogens, and biogenic amines. Each detection principle and component is comprehensively covered and explained. Moreover, the similarities and differences among different types of food contaminants are summarized, aiming to inspire future researchers. Finally, this review concludes with a discussion of the prospects for incorporating AIEgens more effectively into the detection of food contaminants.

Effects of Age on the Biological Properties of Cryopreserved Adipose-Derived Stem Cells and ASC-Enriched Fat Grafts.

BACKGROUND: Donor age is an important factor affecting the biological characteristics of human adipose-derived stem cells. The aim of this study was to compare the effects of age on the biological properties of cryopreserved adipose-derived stem cells and fat survival of cell-assisted lipotransfer. METHODS: Human lipoaspirates were obtained from 60 healthy female patients (aged 18-65 years) who underwent abdominal liposuction. Samples were divided into three groups according to donor age: group A, 18-29 years; group B, 30-49 years; and group C, 50-65 years. Adipose-derived stem cells were obtained by in vitro culture at the second passage and cryopreserved for 4 weeks. The cryopreserved ASCs were examined for biological characteristics, including cell proliferation, wound healing and adipogenic differentiation. Then, the fat survival of cryopreserved ASC-assisted fat transplantation was compared at different ages. RESULTS: SVF viability decreased with increasing age. Moreover, there was a decline in cell proliferation and migration of ASCs with increasing age. A significant difference was found in the adipogenic differentiation of ASCs in the three groups. There were significant differences in graft retention in different age groups. ASC-assisted fat grafting was more effective in young people than in elderly people. CONCLUSIONS: Honor age affects the proliferation and migration of adipose-derived stem cells but not the adipogenic differentiation potential of ASCs. Cryopreserved ASCs from younger people more effectively improved the fat survival of grafts. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Long-term oncologic safety of immediate reconstructive surgery in patients with invasive breast cancer: a retrospective matched-cohort study.

OBJECTIVE: Immediate reconstruction (IR) is a safe and effective surgical treatment for patients with breast cancer. We aimed to assess the prognosis, aesthetic outcomes, and patient satisfaction of IR compared with breast conservation surgery (BCS) and total mastectomy (TM). METHODS: This retrospective matched-cohort study was conducted between May 2005 and December 2014. We established two cohorts according to the tumor (T) size of breast cancer. In the T≤3cm group, cases (IR) and controls (BCS or TM) were matched for age, pathological tumor size, and pathologic nodal status in a 1:1:1 ratio. In the T>3cm group, cases (IR) and controls (TM) were matched with the same factors and ratio. The primary outcome was the 5-year disease-free survival (DFS). The secondary outcome was patient satisfaction and quality of life. RESULTS: A total of 12,678 breast cancer patients were assessed for eligibility, of which 587 were included (T≤3 cm group: 155 IR vs 155 BCS vs 155 TM; T>3cm group: 61 IR vs 61 TM). In the T≤3 cm cohort, patients who underwent IR had no difference compared with those who underwent BCS or TM regarding the 5-year DFS (P=0.539); however, an improved aesthetic satisfaction, psychosocial, and sexual well-being were achieved in the IR group (P<0.001). In the T>3 cm cohort, the IR group had a worse median 5-year DFS (P=0.044), especially for Her2+ or triple-negative breast carcinoma (TNBC) subtypes compared with the TM group. CONCLUSIONS: IR improves aesthetic satisfaction, psychosocial, and sexual well-being for breast cancer patients with T≤3 cm. For patients with T > 3 cm invasive breast cancer, TM is superior to IR as it predicts a better 5-year DFS.

A Security-Enhanced Image Communication Scheme Using Cellular Neural Network.

In the current network and big data environment, the secure transmission of digital images is facing huge challenges. The use of some methodologies in artificial intelligence to enhance its security is extremely cutting-edge and also a development trend. To this end, this paper proposes a security-enhanced image communication scheme based on cellular neural network (CNN) under cryptanalysis. First, the complex characteristics of CNN are used to create pseudorandom sequences for image encryption. Then, a plain image is sequentially confused, permuted and diffused to get the cipher image by these CNN-based sequences. Based on cryptanalysis theory, a security-enhanced algorithm structure and relevant steps are detailed. Theoretical analysis and experimental results both demonstrate its safety performance. Moreover, the structure of image cipher can effectively resist various common attacks in cryptography. Therefore, the image communication scheme based on CNN proposed in this paper is a competitive security technology method.

High-performance and ultraflexible PEDOT/silver nanowires/graphene films for electrochromic applications.

We fabricate a kind of flexible electrochromic (EC) film by spraying the mixed dispersion of silver nanowires (AgNWs) and poly (3, 4-ethylenedioxythiophene) (PEDOT) on the graphene (GR) electrode. AgNWs are embedded in the PEDOT nanoparticles, forming an interlaced conductive network and double electron transport channels; therefore, the disadvantages of poor conductive GR electrodes have been remedied effectively. The subsequent GR-based PEDOT/AgNWs composite films have revealed remarkable optical contrast (63%), high coloration efficiency (${182.8}\;{{\rm cm}^{2}}\;{{\rm C}^{ - 1}}$182.8cm2C-1), and good cycle stability (keeping the optical contrast about 60% after switching cycles of 16,000 s). In addition, the GR-based composite films can keep good EC performance after 2000 cycles of bending tests, while those of the ITO/PET-based composite films decrease dramatically. The ultraflexible GR-based PEDOT/AgNWs composite films with excellent EC properties present an opportunity for fabricating large-area flexible EC devices.

Plasmonic enzyme immunoassay via nanobody-driven controllable aggregation of gold nanoparticles for detection of ochratoxin A in pepper.

Ochratoxin A (OTA) in food poses a serious challenge to public health. Herein, using the nanobody-driven controllable aggregation of gold nanoparticles (AuNPs) in a glucose oxidase-tyramine-horseradish peroxidase (GOx-TYR-HRP) system, we propose a direct competitive plasmonic enzyme immunoassay (dc-PEIA) for OTA detection. The OTA-GOx conjugate catalyzes glucose to produce hydrogen peroxide (H2O2), and then HRP catalyzes H2O2 to generate hydroxyl radical which induces the crosslink of TYR. Crosslinked TYR leads to aggregation of AuNPs through strong electrostatic interactions, which is tunable based on the competition of OTA-GOx and free OTA for binding the immobilized nanobody. The optimized dc-PEIA achieves an instrumental limit of detection (LOD) of 0.275 ng/mL and a visual LOD of 1.56 ng/mL. It exhibits good selectivity for OTA and accuracy in the analysis of pepper samples, with the confirmation of high-performance liquid chromatography. Overall, the dc-PEIA is demonstrated as a useful tool for detecting OTA in food.

Aramid nanofiber membrane decorated with monodispersed silver nanoparticles as robust and flexible SERS chips for trace detection of multiple toxic substances.

Aramid nanofibers (ANFs) as an innovative nanoscale building block exhibit great potential for novel high-performance multifunctional membranes attributed to their extraordinary performance. However, the application of aramid nanofibers in the field of surface enhanced Raman scattering (SERS) sensing has been rarely reported. In this work, aramid nanofibers derived from commercial Kevlar fibers were synthesized by a facile dimethyl sulfoxide/potassium hydroxide (DMSO/KOH) solution treatment. The monodispersed silver nanoparticle-decorated aramid nanofiber (m-Ag@ANF) membranes were constructed by an efficient vacuum filtration technique. Taking advantages of unique intrinsic properties of ANF, the m-Ag@ANF substrates exhibit good flexibility, excellent mechanical properties and prominent thermal stability. Besides, due to the abundance of positively charged amino-group on the ANF substrates, the negatively charged m-AgNPs were uniformly and firmly deposited on the surface of ANF substrate through electrostatic interactions. As a result, the optimal flexible m-Ag-9@ANF SERS substrate exhibits high sensitivity of 10-9 M for methylene blue (MB) and excellent signal reproducibility (RSD = 6.37 %), as well as outstanding signal stability (up to 15 days). Besides, the 2D Raman mapping and FDTD simulations further reveal prominent signal homogeneity and strong electric field distribution for flexible m-Ag-9@ANF SERS substrate. Finally, it is demonstrated that the flexible m-Ag-9@ANF SERS substrate can also be used for detection of toxic molecules on irregular surfaces by a feasible paste-and-read process. The m-Ag@ANF paper exhibits potential applications as a flexible, low-cost, robust and stable SERS sensing platform for trace detection of toxic materials.

Shark anti-idiotypic variable new antigen receptor specific for an alpaca nanobody: Exploration of a nontoxic substitute to ochratoxin A in immunoassay.

Nontoxic substitutes to mycotoxins can facilitate the development of eco-friendly immunoassays. To explore a novel nontoxic substitute to ochratoxin A (OTA), this study screened shark anti-idiotypic variable new antigen receptors (VNARs) against the alpaca anti-OTA nanobody Nb28 through phage display. After four rounds of biopanning of a naïve VNAR phage display library derived from six adult Chiloscyllium plagiosum sharks, one positive clone, namely, P-3, was validated through a phage enzyme-linked immunosorbent assay (phage ELISA). The recombinant anti-idiotypic VNAR AId-V3 was obtained by prokaryotic expression, and the interactions between Nb28 and AId-V3 were investigated via computer-assisted simulation. The affinity of AId-V3 for Nb28 and its heptamer Nb28-C4bpα was measured using Biacore assay. Combining Nb28-C4bpα with AId-V3, a novel direct competitive ELISA (dcELISA) was developed for OTA analysis, with a limit of detection of 0.44 ng/mL and a linear range of 1.77-32.25 ng/mL. The good selectivity, reliability, and precision of dcELISA were confirmed via cross-reaction analysis and recovery experiments. Seven commercial pepper powder samples were tested using dcELISA and validated using high-performance liquid chromatography. Overall, the shark anti-idiotypic VNAR was demonstrated as a promising nontoxic substitute to OTA, and the proposed method was confirmed as a reliable tool for detecting OTA in food.

Ready-to-use ratiometric bioluminescence immunosensor for detection of ochratoxin a in pepper.

Rapid, portable, and accurate detection tools for monitoring ochratoxin A (OTA) in food are essential for the guarantee of food safety and human health. Herein, as a proof-of-concept, this study proposed a ratiometric bioluminescence immunosensor (RBL-immunosensor) for homogeneous detection of OTA in pepper. The construct of the RBL-immunosensor consists of three components, including the large fragment of the split nanoluciferase (NanoLuc)-tagged nanobody (NLg), the small fragment of the split NanoLuc-tagged mimotope peptide heptamer (MPSm), and the calibrator luciferase (GeNL). The specific nanobody-mimotope peptide interaction between NLg and MPSm induces the reconstitution of the NanoLuc, which catalyzes the Nano-Glo substrate and produces a blue emission peak at 458 nm. Meanwhile, GeNL can produce a green emission peak at 518 nm upon substrate conversion via bioluminescent resonance energy transfer (BRET). Therefore, the concentration of OTA can be linked to the variation of the bioluminescence signal (λ458/λ518) measured by microplate reader and the variation of the blue/green ratio measured by smartphone via the competitive immunoreaction where OTA competes with MPSm to bind NLg. The immunosensor is ready-to-use and works by simply mixing the components in a one-step incubation of 10 min for readout. It has a limit of detection (LOD) of 0.98 ng/mL by a microplate reader and an LOD of 1.89 ng/mL by a smartphone. Good selectivity and accuracy were confirmed for the immunosensor by cross-reaction analysis and recovery experiments. The contents of OTA in 10 commercial pepper powder samples were tested by the RBL-immunosensor and validated by high-performance liquid chromatography. Hence, the ready-to-use RBL-immunosensor was demonstrated as a highly reliable tool for detection of OTA in food.

Au@CuS Nanoshells for Surface-Enhanced Raman Scattering Image-Guided Tumor Photothermal Therapy with Accelerated Hepatobiliary Excretion.

Gold-based nanoparticles for surface-enhanced Raman scattering (SERS) imaging show great potential for precise tumor detection and photothermal therapy (PTT). However, the metabolizability of gold nanoparticles (Au NPs) raises big concerns. Herein, we designed a core-shelled nanostructure of copper sulfide (CuS)-coated Au NPs with surface pegylation (PEG-Au@CuS NSs). The excreted Au in the gallbladders at 1 h and 4 h in mice injected with PEG-Au@CuS NSs was 8.2- and 19.1-fold of that with the pegylated Au NPs (PEG-AuNPs) of the same Au particle size, respectively. By loading the Raman reporter 3,3'-Diethylthiatricarbocyanine iodide (DTTC) in the core-shell junction of PEG-Au@CuS NSs, the PEG-Au-DTTC@CuS NSs exhibited the Raman signal-to-noise (S/N) ratio of 4.01 after 24 h of intravenous (IV) injection in the mice bearing an orthotopic CT26-Luc colon tumor. By contrast, the DTTC-coated PEG-AuNPs (PEG-Au-DTTC NPs) achieved an S/N ratio of 2.71. Moreover, PEG-Au-DTTC@CuS NSs exhibited an increased photothermal conversion effect compared with PEG-Au-DTTC NPs excited with an 808-nm laser. PEG-Au-DTTC@CuS NSs enabled intraoperative SERS image-guided photothermal therapy for a complete cure of the colon tumor-bearing mice. Our data demonstrated that the PEG-Au-DTTC@CuS NSs are promising intraoperative Raman image-guided theranostic nanoplatform with enhanced hepatobiliary excretion.

Lateral Flow Immunochromatographic Assay for Competitive Detection of Crustacean Allergen Tropomyosin Using Phage-Displayed Shark Single-Domain Antibody.

The common food allergy crustacean tropomyosin (TM) poses a significant food safety challenge, which requires rapid and sensitive methods for screening TM in food. Herein, the variable new antigen receptor (VNAR) single-domain antibodies specific for the crustacean TM were isolated from a naïve phage-displayed shark VNAR library. Subsequently, a lateral flow immunochromatographic assay (LFIA) based on the gold nanoparticle-labeled phage-displayed shark VNAR (AuNPs@PSV) probe was developed for the detection of TM in food. The AuNPs@PSV-LFIA took 15 min for one test and had a visual limit of detection (vLOD) of 0.1 µg/mL and an instrumental LOD of 0.02 µg/mL. Good selectivity, accuracy, precision, and stability were confirmed for the AuNPs@PSV-LFIA. Moreover, the test results of 21 commercially available food products consisted of the allergen labels and were validated by a commercial ELISA kit. Therefore, this work demonstrated the great potential of VNAR for detecting TM in food by LFIA.

Colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor using phage-displayed shark nanobody for the detection of crustacean allergen tropomyosin.

Tropomyosin (TM) is the primary allergenic protein responsible for crustacean food allergies, and thus sensitive and rapid methods are required for the screening of crustacean TM in food. In this study, using the phage-displayed shark nanobody (PSN) as a multifunctional biomaterial, we developed a colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor (CM/SERS-LFI) for competitive detection of crustacean TM. The SERS tag AuMBA@AgNPs with the Raman signal molecule 4-mercaptobenzoic acid (4-MBA) was prepared and immobilized on the PSN to construct the immunoprobe AuMBA@Ag-PSN. The probe can identify free TM that competes with TM on the T-line, and the optimized CM/SERS-LFI enables quantitative analysis of TM using the probe with a limit of detection (LOD) of 0.0026 µg/mL (SERS mode) and 0.0057 µg/mL (colorimetric mode), respectively. Additionally, it can implement a qualitative analysis by the naked eye with a visual LOD of 0.01 µg/mL. The CM/SERS-LFI exhibited excellent performance in the tests of selectivity, accuracy, precision, and stability. Moreover, the method's effectiveness in the analysis of real samples was confirmed by a commercial ELISA kit. Therefore, the developed CM/SERS-LFI was demonstrated to be a powerful and reliable tool for the rapid and sensitive detection of crustacean TM in food.

Flexible, scalable and simple-fabricated silver nanorod-decorated bacterial nanocellulose SERS substrates cooperated with portable Raman spectrometer for on-site detection of pesticide residues.

Owing to good flexibility, prominent mechanical properties, three-dimensional (3D) nanofibrous structure and low background interference, sustainable bacterial nanocellulose (BNC) is a highly attractive matrix material for surface-enhanced Raman scattering (SERS) sensor. Herein, a highly sensitive, flexible and scalable silver nanorod-decorated BNC (AgNRs@BNC) SERS sensor is developed by a simple vacuum-assisted filtration. The AgNRs were firmly locked in the 3D nanofibrous network of cellulose nanofibers upon vacuum drying process, resulting in the formation of 3D SERS hotspots with a depth of more than 10 µm on the sensor. With 4-aminothiophenol (4-ATP) as a target molecule, a lowest distinguishable level of 10-12 M and a high enhancement factor of 1.1 × 109 were realized by the optimal AgNRs1.5@BNC SERS sensor. Moreover, the AgNRs@BNC SERS sensor exhibits high detectable level of 10-9 M for thiram molecules by integrating with a portable Raman spectrometer. Besides, toxic thiram residues on grape surface could be directly on-site identified by the combination of AgNRs@BNC SERS sensors and a portable Raman spectrometer through a feasible press-and-peel method. The flexible AgNRs@BNC SERS sensor cooperated with portable Raman system demonstrates great potential for on-site detection of pesticide residues on irregular food surfaces.

Colorimetric and chemiluminescent enzyme immunoassays based on the alkaline phosphatase-tagged single-chain variable fragment fusion tracer for detecting zearalenone in agro-products.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin and seriously threatens food safety, which requires rapid and sensitive detection methods for monitoring ZEN in agro-products. Herein, an alkaline phosphatase-tagged single-chain variable fragment fusion protein (ALP-scFv) was used as a bifunctional tracer to develop a colorimetric enzyme immunoassay (CEIA) and a chemiluminescent enzyme immunoassay (CLEIA) for ZEN. In addition, the interactions between scFv and ZEN were exploited by computer-assisted simulation, and four key amino acid sites were preliminarily identified. After optimization, the CEIA and CLEIA exhibited a limit of detection of 0.02 and 0.006 ng/mL, respectively. Furthermore, both methods showed favorable accuracy in recovery experiments and good selectivity in cross reactions. Moreover, the detection results of the actual samples from both methods correlated well with those from high-performance liquid chromatography. Overall, the ALP-scFv fusion tracer-based CEIA and CLEIA are demonstrated as reliable tools for ZEN detection in food.

Self-stacked small molecules for ultrasensitive, substrate-free Raman imaging in vivo.

Raman spectroscopy using surface-enhanced Raman scattering (SERS) nanoprobes represents an ultrasensitive and high-precision technique for in vivo imaging. Clinical translation of SERS nanoprobes has been hampered by biosafety concerns about the metal substrates used to enhance Raman signals. We report a set of small molecules with bis-thienyl-substituted benzobisthiadiazole structures that enhance Raman signal through self-stacking rather than external substrates. In our technique, called stacking-induced charge transfer-enhanced Raman scattering (SICTERS), the self-stacked small molecules form an ordered spatial arrangement that enables three-dimensional charge transfer between neighboring molecules. The Raman scattering cross-section of SICTERS nanoprobes is 1350 times higher than that of conventional SERS gold nanoprobes of similar particle size. SICTERS outperforms SERS in terms of in vivo imaging sensitivity, resolution and depth. SICTERS is capable of noninvasive Raman imaging of blood and lymphatic vasculatures, which has not been achieved by SERS. SICTERS represents an alternative technique to enhance Raman scattering for guiding the design of ultrasensitive substrate-free Raman imaging probes.

Intravital Microscopy Reveals Endothelial Transcytosis Contributing to Significant Tumor Accumulation of Albumin Nanoparticles.

The principle of enhanced permeability and retention (EPR) effect has been used to design anti-cancer nanomedicines over decades. However, it is being challenged due to the poor clinical outcome of nanoparticles and controversial physiological foundation. Herein, we use a near-infrared-II (1000-1700 nm, NIR-II) fluorescence probe BPBBT to investigate the pathway for the entry of human serum albumin-bound nanoparticles (BPBBT-HSA NPs) into tumor compared with BPBBT micelles with phospholipid-poly (ethylene glycol) of the similar particle size about 110 nm. The plasma elimination half-life of BPBBT micelles was 2.8-fold of that of BPBBT-HSA NPs. However, the area under the BPBBT concentration in tumor-time curve to 48 h post-injection (AUCtumor0→48h) of BPBBT-HSA NPs was 7.2-fold of that of BPBBT micelles. The intravital NIR-II fluorescence microscopy revealed that BPBBT-HSA NPs but not BPBBT micelles were transported from the tumor vasculature into tumor parenchyma with high efficiency, and endocytosed by the tumor cells within 3 h post-injection in vivo. This effect was blocked by cross-linking BPBBT-HSA NPs to denature HSA, resulting in the AUCtumor0→48h decreased to 22% of that of BPBBT-HSA NPs. Our results demonstrated that the active process of endothelial transcytosis is the dominant pathway for albumin-bound nanoparticles' entry into tumor.