Research Progress of Cyanine-based Near-Infrared Fluorescent Probes for Biological Application.

Cyanine-based near-infrared (NIR) fluorescent probes have played vital roles in biological application due to their low interference from background fluorescence, deep tissue penetration, high sensitivity, and minimal photodamage to biological samples. They are widely utilized in molecular recognition, medical diagnosis, biomolecular detection, and biological imaging. Herein, we provide a review of recent advancements in cyanine-based NIR fluorescent probes for the detection of pH, cells, tumor as well as their application in photothermal therapy (PTT) and photodynamic therapy (PDT).

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Chemoselective Fluorogenic Bioconjugation of Vicinal Dithiol-Containing Proteins for Live Cellular Imaging via Small Molecular Conjugate Acceptors.

To develop small molecular fluorogenic tools for the chemoselective labeling of vicinal dithiol-containing proteins (VDPs) in live cells is important for studying intracellular redox homeostasis. With this research, we developed small molecule-based fluorescent probes, achieving selective labeling of VDPs through thiol-thiol substitutions on bisvinylogous thioester conjugated acceptors (IDAs). Initially, IDAs demonstrated its ability to bridge vicinal cysteine-sulfhydryls on a peptide as a mimic. Then, the peptide complex could be decoupled to recover the original peptide-SH in the presence of dithiothreitol. Furthermore, fluorometric signal amplification of the fluorescent probes occurred with high sensitivity, low limit of detection, and selectivity toward vicinal dithiols on reduced bovine serum albumin, as an example of real world VDPs. More importantly, the probes were utilized successfully for labeling of endogenous VDPs at different redox states in live cells. Thus, the bisvinylogous thioester-based receptor as a functional probe represents a new platform for uncovering the function of VDPs in live cells.

A HBDI-Based Fluorescent Probe for Labeling Endoplasmic Reticulum in Living Cells.

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells and is closely involved in the synthesis and processing of proteins, as well as the storage, regulation, and release of calcium. A series of signaling pathways within the ER play a crucial part in the pathogenesis of various diseases, including cancer. Thus, it is necessary to design ER-targeting probes to monitor these signaling pathways. Additionally, precision medicine also requires new ER-targeting group to facilitate the delivery of drug cargoes to the ER. However, only a limited number of ER-targeting groups have been used for the design of fluorescent probes for ER imaging in living cells, as well as the development of ER-targeted drug delivery systems. Herein, a new ER-targeting fluorescent probe (BDI-ER) was designed and prepared. BDI-ER contains the hydrophilic fluorophore, derived from the core structure of GFP, and two hydrophobic octadecane chains. The amphipathic nature of BDI-ER facilitates localization in the ER. Live cell imaging demonstrated selective localization of BDI-ER towards ER compared to other organelles. Additionally, co-localization imaging in various cell lines indicate that BDI-ER is effective at targeting the ER.

Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5'-triphosphate in Cellular Applications.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO-) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO- and ATP. ONOO- selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO- and ATP products, ATP-LW allows ONOO- levels to be monitored in the green channel (λex = 488 nm, λem = 500-575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575-650 nm). The use of ATP-LW as a combined ONOO- and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO- donor).

Molecularly imprinted photoelectrochemical sensing based on ZnO/polypyrrole nanocomposites for acrylamide detection.

A highly sensitive quenching molecular imprinting (MIP) photoelectrochemical (PEC) sensor was proposed to detect acrylamide (AM) by using the photoactive composite of ZnO and polypyrrole (PPy) as the PEC signal probe. ZnO, with high electron mobility, excellent chemical and thermal stability as well as good biocompatibility, was selected as the photoelectrically active material. A polypyrrole film was formed on the nanodisk ZnO by electrochemical polymerization, and the recognition site of AM was left on the surface of the PPy film by elution, enabling the specific detection of AM. The transfer of electrons will be hindered when AM is adsorbed on the ZnO/PPy nanocomposites surface, which results in the decrease of photocurrent signal. The proposed molecularly imprinted PEC sensor exhibits significant detection performance of AM in the range of 10-1 M-2.5 × 10-9 M with a LOD of 2.147 × 10-9 M (S/N = 3). The use of photoelectrochemical technology combined with molecular imprinting technology enables the PEC sensor to have excellent selectivity, superior repeatability, preferable stability, low cost, and easy construction, providing a new method for the detection of AM. The high recovery rate in the detection of real samples of potato chips and biscuits indicates that the proposed PEC sensor has potential in monitoring the emerging food safety risks.

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents.

In this tutorial review, we will explore recent advances in the construction and application of Förster resonance energy transfer (FRET)-based small-molecule fluorescent probes. The advantages of FRET-based fluorescent probes include: a large Stokes shift, ratiometric sensing and dual/multi-analyte responsive systems. We discuss the underlying energy donor-acceptor dye combinations and emphasise their applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvionments and dual/multi-analyte responsive systems.

Ultrasmall Au-Ag Alloy Nanoparticles: Protein-Directed Synthesis, Biocompatibility, and X-ray Computed Tomography Imaging.

The ultrasmall sizes of nanoparticles have attracted significant attention for potential applications in the fields of catalysis and nanomedicine. Herein, we reported on the green preparation and X-ray computed tomography (CT) imaging of ultrasmall bimetallic bovine serum albumin-directed gold-silver (Au-Ag@BSA) nanoparticles (2-4 nm) using BSA as a stabilizing and template-directed agent. Further, the effects of synthesis conditions were systematically explored to prepare products by adjusting the different molar ratios of Au/Ag. The resulting Au-Ag@BSA nanoparticles exhibited the spherical shape, well-dispersed ability, as well as long-term room-temperature stability. The cytotoxicity effects of Au-Ag@BSA nanoparticles on A549 and MCF-7 cells were compared with those of individual Ag nanoparticles, and the results indicated a lower cytotoxicity effect by Au-Ag@BSA nanoparticles. Furthermore, the in vivo toxicity of Au-Ag@BSA nanoparticles was investigated in the early stage zebrafish embryos. The results indicate that there are not any obvious changes of survival and hatching percentages at multiple growth stages (4-120 hpf) even with a high level of Au-Ag@BSA nanoparticles (up to 80 mM), revealing good biocompatibility. Interestingly, a rational design of the Au/Ag molar ratio (3:2) surprisingly possessed enhanced CT performance compared to the performance of the Au nanoparticles and iohexol. Accordingly, this study highlights a new prospect in the green preparation of ultrasmall alloy nanomaterials with good biocompatibility and will be of great interest in developing CT contrast agent, catalyst, as well as drug delivery carrier.

3D carbon nanosphere and gold nanoparticle-based voltammetric cytosensor for cell line A549 and for early diagnosis of non-small cell lung cancer cells.

An electrochemical cytosensor for the detection of the non-small-cell lung cancer cell line A549 (NSCLC) had been developed. A microwave-hydrothermal method was employed to prepare monodisperse colloidal carbon nanospheres (CNSs). Gold nanoparticles (AuNPs) were placed on the surface of the colloidal CNSs by self-assembly to obtain 3D-structured microspheres of the type CNS@AuNP. The results of an MTT assay show the microspheres to possess good biocompatibility. The CNS@AuNP nanocomposite was then placed, in a chitosan film, on a glassy carbon electrode (GCE). The voltammetric signals and detection sensitivity are significantly enhanced owing to the synergistic effect of CNSs and AuNPs. A cytosensor was then obtained by immobilization of antibody against the carcinoembryonic antigen (which is a biomarker for NSCLC) on the GCE via crosslinking with glutaraldehyde. Hexacyanoferrate is used as an electrochemical probe, and the typical working voltage is 0.2 V (vs. SCE). If exposed to A549 cells, the differential pulse voltammetric signal decreases in the 4.2 × 10-1 to 4.2 × 10-6 cells mL-1 concentration range, and the detection limit is 14 cells mL-1 (at S/N = 3). Graphical abstract Schematic presentation of design strategy and fabrication process of the electrochemical cytosensor for A549 cells. (CNS: carbon nanospheres; GA: glutaraldehyde; PEI: polyethyleneimine; AuNPs: gold nanoparticles; BSA: Bovine serum albumin).

A renewable test strip combined with solid-state ratiometric fluorescence emission spectra for the highly selective and fast determination of hydrazine gas.

N2H4 is one of the most toxic and explosive chemicals. In particular, N2H4 vapor is dangerously unstable. However, the determination of N2H4 gas has lagged behind the detection of N2H4 in aqueous solution or biological fluid in terms of the crucial ability to discriminate and quantify N2H4. Despite the multitude of fluorescent probe-coated test strips that have been used for N2H4 gas detection, these techniques mainly rely on the observation by eye of fluorescence color changes, which will hamper the discrimination of subtle color changes on the test strips due to the limited capacity of the eye to perceive and differentiate colors. Herein, we developed a new technique combining a renewable test strip and solid-state ratiometric fluorescence readout. The results confirmed that the prepared test strips could be used as a solid-state fluorescence sensor for the fast capture (within 5 min) and quantification of N2H4 gas. Additionally, the test strips could be easily renewable. Thus, this test strip-based approach provides an efficient tool for N2H4 gas detection, which is particularly significant for the real-time monitoring of N2H4 gas in airports and the environmental atmosphere in chemical plant regions. This design concept presents a new avenue for developing solid-state fluorescence sensors for gas detection.

Hyaluronic acid-grafted three-dimensional MWCNT array as biosensing interface for chronocoulometric detection and fluorometric imaging of CD44-overexpressing cancer cells.

A sandwich-type electrochemical cytosensor is described for quantitative determination of CD44-overexpressing HeLa cells. Hyaluronic acid (HA) acts as a targeting molecule that was in-situ incorporated into the sensor based on the use of an indium tin oxide (ITO) electrode modified with multi-walled carbon nanotubes (MWCNTs). The 3D-MWCNT structure is shown to strongly improve the electronic properties and surface chemical reactivities. The HA-modified sensor exhibits a highly sensitive response to HeLa cells. A sandwiched hybridization protocol was then established using BIO [an N-butyl-4-(6'-aminohexyl)amino-1,8-naphthalimide probe modified with HA] as the tracing labels of the fluorescent probes for targeting CD44-positive tumor cells. The signal amplification was thereby maximized and measured by chronocoulometry. The binding of CD44-positive HeLa cells to the HA modified sensing layer causes a decrease in chronocoulometric response. The signal decreases linearly in the 2.1 × 102 to 2.1 × 107 HeLa cells·mL-1 concentration range with a detection limit of 70 cells·mL-1. Such a sandwich-type assay may be tailored as a sensitive candidate for detecting low levels of tumor cells. Graphical abstract Schematic of a sandwich cytosensor based on hyaluronic acid-grafted 3D-MWCNT as biosensing interface and BIO as fluorescent probe. This biosensor possessed excellent electrochemical activity, high sensitivity and selectivity, providing a dynamical tracking and detecting platform for CD44-positive tumor cells.

Gemcitabine-loaded gold nanospheres mediated by albumin for enhanced anti-tumor activity combining with CT imaging.

Currently, the development of a more effective therapy approach overcoming the resistance to chemotherapy for lung cancer still faces great challenges and requires further investigation. Herein, we fabricated gemcitabine (Gem) loaded gold nanospheres mediated by bovine serum albumin (Au@BSA) for the efficient delivery of therapeutic agents into the human pulmonary carcinoma cells (A549) and simultaneous computed tomography (CT) imaging. These nanoparticles (Au@BSA) with an approximate diameter of 10 nm were single crystals and prepared through a protein-directed facile one-pot synthesis via the reduction of chloroauric acid (HAuCl4) utilizing bovine serum albumin (BSA) as the stabilizer. The resulting positive charged Au@BSA nanospheres were conjugated with the negative Gem which served as the chemotherapeutic payload by electrostatic layer-by-layer assembling interactions to form Au@BSA-Gem. Our results demonstrated that the obtained biomimetic Au@BSA nanospheres were biocompatible with low cytotoxicity while the Gem-loaded ones exhibited obvious effects with efficiencies significantly greater than that of the free Gem.

Electrochemiluminescence evaluation for carbohydrate antigen 15-3 based on the dual-amplification of ferrocene derivative and Pt/BSA core/shell nanospheres.

Herein, a novel methodology for ultrasensitive and facile breast cancer biomarker carbohydrate antigen 15-3 (CA15-3) evaluation was proposed by fabricating a sandwiched ECL immunosensor. In the protocol, MOCs-Fc and Pt@BSA-luminol nanohybrids were successfully synthesized and further employed to achieve dual-amplification strategy for luminol-H2O2 system. Notably, inherent porous microstructure and large specific surface area from MOCs enabled a high loading of Fc, which succeeded in catalyzing luminol-H2O2 ECL emission and therefore enhancing ECL response. In addition, higher sensitivity could be realized due to the excellent electronic conductivity of MOCs. Furthermore, the as-obtained Pt@BSA- luminol was not only employed as capture probe to recognize CA15-3 after hybridization with Ab2, but also played a crucial role in acting as ECL signal probe due to the presence of massive luminol. It is of vital importance that Pt@BSA core/shell nanospheres showed admirable catalytic effect towards H2O2, which resulted in more excited state luminol and stronger ECL intensity. Therefore, the synergistic amplification strategy of MOCs-Fc and Pt@BSA nanohybrids offered an extremely enhanced ECL signal. The well-established applicable ECL immune- sensing platform displayed favorable analytical performance for CA15-3. In summary, the proposed ECL immunosensor opened a new era for sensitive CA15-3 evaluation and offered a promising platform for clinical breast cancer diagnostics.

Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells.

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

A biomimetic Au@BSA-DTA nanocomposites-based contrast agent for computed tomography imaging.

Early detection of cancer is increasingly important for being considered to increase the survival rate in the treatment process. The past decades years have witnessed the great progress in the biological detection application of gold nanoparticles. Herein, we reported a facile one-pot synthesis process to obtain gold nanoparticles (Au@BSA) with bovine serum albumin (BSA) as a biotemplate following with conjugation of diatrizoic acid (DTA) for a potential X-ray computed tomography (CT) imaging contrast agent (Au@BSA-DTA). The as-prepared biomimetic material was characterized systematically by several techniques. It was shown that the prepared biomaterial is colloid stable under the tested range of pH and temperature. The cell cytotoxicity assay, hemolytic assay and cell morphology observation showed that Au@BSA-DTA has good biocompatibility and hemocompatibility at a concentration of Au even up to 80µg/mL. Besides, the biomimetic material Au@BSA-DTA with double radiodense elements of Au and iodine displayed much stronger CT imaging effect compared with the traditional small molecule contrast agents, which paves the potential clinical application in cancer early diagnosis.

Functionalized graphene oxide/Fe3O4 hybrids for cellular magnetic resonance imaging and fluorescence labeling.

In this work, we developed a T2-weighted contrast agent based on graphene oxide (GO)/Fe3O4 hybrids for efficient cellular magnetic resonance imaging (MRI). The GO/Fe3O4 hybrids were obtained by combining with co-precipitation method and pyrolysis method. The structural, surface and magnetic characteristics of the hybrids were systematically characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), AFM, Raman, FT-IR and XRD. The GO/Fe3O4 hybrids were functionalized by modifying with anionic and cationic polyelectrolyte through layer-by-layer assembling. The fluorescence probe fluorescein isothiocyanate (FITC) was further loaded on the surface of functionalized GO/Fe3O4 hybrids to trace the location of GO/Fe3O4 hybrids in cells. Functionalized GO/Fe3O4 hybrids possess good hydrophilicity, less cytotoxicity, high MRI enhancement with the relaxivity (r2) of 493mM-1s-1 as well as cellular MRI contrast effect. These obtained results indicated that the functionalized GO/Fe3O4 hybrids could have great potential to be utilized as cellular MRI contrast agents for tumor early diagnosis and monitoring.

An ultrasensitive multi-walled carbon nanotube-platinum-luminol nanocomposite-based electrochemiluminescence immunosensor.

An ultrasensitive electrochemiluminescence (ECL) immunosensor for carbohydrate antigen 19-9 (CA19-9) detection using multi-walled carbon nanotube-platinum-luminol nanocomposites (MWCNT-Pt-luminol) as nanointerface and signal tags was designed. First, the MWCNT-Pt-luminol nanocomposite was decorated on the surface of a glassy carbon electrode (GCE) through the film-forming properties of chitosan (Chi). Then, the CA19-9 antibody (anti-CA19-9) was attached to the modified electrode surface via crosslinking with glutaraldehyde (GA). When CA19-9 was captured by its antibody immobilized on the immune platform via immunoreaction, the ECL signal intensity decreased. Under optimal conditions, the proposed ECL immunosensor showed excellent performance for CA19-9 ranging from 0.0001 U mL-1 to 10.0 U mL-1, with a detection limit of 0.000046 U mL-1 (S/N = 3) and a correlation coefficient of R = 0.9980. This is attributed to the fact that the MWCNTs-Pt nanomaterial has excellent conductivity and it could facilitate the decomposition of H2O2 to generate various reactive oxygen species (ROSs); thus, the ECL signals of luminol were effectively amplified and the sensitivity of the sensor was greatly increased. The prepared ECL immunosensor displayed simple, fast analysis, excellent stability, good reproducibility, and high specificity. Moreover, the developed ECL immunosensor provided satisfactory results in the determination of CA19-9 in real human serum samples.

A MWCNTs-Pt nanohybrids-based highly sensitive electrochemiluminescence sensor for flavonoids assay.

Electrochemiluminescent (ECL) assay has gradually drawn increasing interest to be considered as one of the potential strategies to be applicable in a wide range of medicine target determination. In this protocol, a facile and ultrasensitive ECL sensor platform was developed aiming at rapidly quantitative detection of polyphenolic flavonoids based on multiwall carbon nanotubes (MWCNTs)/platinum nanoparticles (PtNPs) nanohybrids/nafion/Ru(bpy)32+ modified glassy carbon electrode (GCE) in Ru(bpy)32+/tri-n-propylamine (TPA) co-reaction system. Attributed to the synergistic ECL signal amplification of both PtNPs and MWCNTs facilitating the electron transfer, this proposed sensor could sensitively analyze the flavonoids through an annihilation ECL emission mechanism since the less generated amount of excitation state Ru(Ⅱ)* on account of the principle that flavones are prone to donate electrons and scavenge free radicals to further show a competitive relationship with Ru(Ⅲ) on the reaction with TPA*. The influences of pH, Ru(bpy)32+ absorption time and TPA co-reactant concentration were investigated and the sensor exhibited a linear response to flavonoids when the concentration ranges from 6.50×10-11 to 1.00×10-7M with a detection limit (LOD) of 2.17×10-11M (S/N=3) upon the optimized conditions. Furthermore, high sensitivity, excellent stability and reproducibility were verified to declare the promise for being applicable to analyze the flavones content in real flavones medicine samples and ginkgo leaves extracts with satisfactory results.

Multiwalled carbon nanotubes/gold nanocomposites-based electrochemiluminescent sensor for sensitive determination of bisphenol A.

An electrochemiluminescence (ECL) sensor for bisphenol A was proposed by using L-cysteine-functionalized multiwalled carbon nanotubes/gold nanocomposites-modified glassy carbon electrode (MWCNTs-Au/GCE) based on ECL of peroxydisulfate solution. The ECL behaviors of peroxydisulfate solution had been investigated at the chitosan/MWCNTs-Au/GCE, and bisphenol A was found to have quenching effects on the ECL of peroxydisulfate solution. Both Au nanoparticles (AuNPs) and multiwalled CNTs could promote the electron transfer and synergetically amplify the ECL signal of peroxydisulfate solution. Under the optimized conditions, the ECL signal intensity was linear with the concentration of bisphenol A in the concentration range between 0.25 and 100 µM (R = 0.9931) with a detection limit (S/N = 3) of 0.083 µM. The constructed ECL sensor has the advantages of simplicity, sensitivity, good selectivity, and reproducibility, exhibiting a great potential application in the determination of bisphenol A.

A Functional CT Contrast Agent for In Vivo Imaging of Tumor Hypoxia.

Hypoxia, which has been well established as a key feature of the tumor microenvironment, significantly influences tumor behavior and treatment response. Therefore, imaging for tumor hypoxia in vivo is warranted. Although some imaging modalities for detecting tumor hypoxia have been developed, such as magnetic resonance imaging, positron emission tomography, and optical imaging, these technologies still have their own specific limitations. As computed tomography (CT) is one of the most useful imaging tools in terms of availability, efficiency, and convenience, the feasibility of using a hypoxia-sensitive nanoprobe (Au@BSA-NHA) for CT imaging of tumor hypoxia is investigated, with emphasis on identifying different levels of hypoxia in two xenografts. The nanoprobe is composed of Au nanoparticles and nitroimidazole moiety which can be electively reduced by nitroreductase under hypoxic condition. In vitro, Au@BSA-NHA attain the higher cellular uptake under hypoxic condition. Attractively, after in vivo administration, Au@BSA-NHA can not only monitor the tumor hypoxic environment with CT enhancement but also detect the hypoxic status by the degree of enhancement in two xenograft tumors with different hypoxic levels. The results demonstrate that Au@BSA-NHA may potentially be used as a sensitive CT imaging agent for detecting tumor hypoxia.