Photoacoustic Probes for Molecular Detection: Recent Advances and Perspectives

Photoacoustic (PA) imaging (PAI) is a noninvasive and nonionizing biomedical imaging modality that combines the advantages of optical imaging and ultrasound imaging. Based on PAI, photoacoustic detection (PAD) is an emerging approach that is involved with the interaction between PA probes and analytes resulting in the changes of photoacoustic signals for molecular detection with rich contrast, high resolution, and deep tissue penetration. This Review focuses on the recent development of PA probes in PAD. The following contents will be discussed in detail: 1) the construction of PA probes; 2) the applications and mechanisms of PAD to different types of analytes, including microenvironments, small biomolecules, or metal ions; 3) the challenges and perspectives of PA probes in PAD.     

Surfactant‐Stripped Micelles for NIR‐II Photoacoustic Imaging through 12 cm of Breast Tissue and Whole Human Breasts

Surfactant‐stripped micelles are formed from a commercially available cyanine fluoroalkylphosphate (CyFaP) salt dye and used for high contrast photoacoustic imaging (PAI) in the second near‐infrared window (NIR‐II). The co‐loading of Coenzyme Q10 into surfactant‐stripped CyFaP (ss‐CyFaP) micelles improves yield, storage stability, and results in a peak absorption wavelength in the NIR‐II window close to the 1064 nm output of Nd‐YAG lasers used for PAI. Aqueous ss‐CyFaP dispersions exhibit intense NIR‐II optical absorption, calculated to be greater than 500 at 1064 nm. ss‐CyFaP is detected through 12 cm of chicken breast tissue with PAI. In preclinical animal models, ss‐CyFaP is visualized in draining lymph nodes of rats through 3.1 cm of overlaid chicken breast tissue. Following intravenous administration, ss‐CyFaP accumulates in neoplastic tissues of mice and rats bearing orthotopic mammary tumors without observation of acute toxic side effects. ss‐CyFaP is imaged through whole compressed human breasts in three female volunteers at depths of 2.6–5.1 cm. Taken together, these data show that ss‐CyFaP is an accessible contrast agent for deep tissue PAI in the NIR‐II window.     

Highly sensitive multiple microRNA detection based on fluorescence quenching of graphene oxide and isothermal strand-displacement polymerase reaction

A simple, highly sensitive, and selective multiple microRNA (miRNA) detection method based on the graphene oxide (GO) fluorescence quenching and isothermal strand-displacement polymerase reaction (ISDPR) was proposed. The capability to discriminate ssDNA and double-stranded nucleic acid structure coupled with the extraordinary fluorescence quenching of GO on multiple organic dye allows the proposed strategy to simultaneously and selectively detect several miRNA labeled with different dyes in the same solution, while the ISDPR amplification endows the detection method with high sensitivity. The strong interaction between ssDNA and GO led to the fluorescent ssDNA probe exhibiting minimal background fluorescence. Upon the recognition of specific target miRNA, an ISDPR was triggered to produce numerous massive specific DNA-miRNA duplex helixes, and a strong emission was observed due to the weak interaction between the DNA-miRNA duplex helix and GO. A miRNA biosensor down to 2.1 fM with a linear range of 4 orders of magnitude was obtained. Furthermore, the large planar surface of GO allows simultaneous quenching of several DNA probes with different dyes and produces a multiple biosensing platform with high sensitivity and selectivity, which has promising application in profiling the pattern of miRNA expression and biomedical research.     

Functionalized NIR‐II Semiconducting Polymer Nanoparticles for Single‐cell to Whole‐Organ Imaging of PSMA‐Positive Prostate Cancer

Development of molecular probes holds great promise for early diagnosis of aggressive prostate cancer. Here, 2‐[3‐(1,3‐dicarboxypropyl) ureido] pentanedioic acid (DUPA)‐conjugated ligand and bis‐isoindigo‐based polymer (BTII) are synthesized to formulate semiconducting polymer nanoparticles (BTII‐DUPA SPN) as a prostate‐specific membrane antigen (PSMA)‐targeted probe for prostate cancer imaging in the NIR‐II window. Insights into the interaction of the imaging probes with the biological targets from single cell to whole organ are obtained by transient absorption (TA) microscopy and photoacoustic (PA) tomography. At single‐cell level, TA microscopy reveals the targeting efficiency, kinetics, and specificity of BTII‐DUPA SPN to PSMA‐positive prostate cancer. At organ level, PA tomographic imaging of BTII‐DUPA SPN in the NIR‐II window demonstrates superior imaging depth and contrast. By intravenous administration, BTII‐DUPA SPN demonstrates selective accumulation and retention in the PSMA‐positive tumor, allowing noninvasive PA detection of PSMA overexpressing prostate tumors in vivo. The distribution of nanoparticles inside the tumor tissue is further analyzed through TA microscopy. These results collectively demonstrate BTII‐DUPA SPN as a promising probe for prostate cancer diagnosis by PA tomography.     

Flexible Photothermal Assemblies with Tunable Gold Patterns for Improved Imaging‐Guided Synergistic Therapy

Self‐assembly of gold nanoparticles demonstrates a promising approach to realize enhanced photoacoustic imaging (PAI) and photothermal therapy (PTT) for accurate diagnosis and efficient cancer therapy. Herein, unique photothermal assemblies with tunable patterns of gold nanoparticles (including arcs, rings, ribbons, and vesicles) on poly(lactic‐co‐glycolic acid) (PLGA) spheres are constructed taking advantage of emulsion‐confined and polymer‐directed self‐assembly strategies. The influencing factors and formation mechanism to produce the assemblies are investigated in details. Both the emulsion structure and migration behaviors of amphiphilic block copolymer tethered gold nanoparticles are found to contribute to the formation of versatile photothermal assemblies. Hyaluronic acid‐modified R‐PLGA‐Au (RPA) exhibits outstanding photothermal performances under NIR laser irradiation, which is induced by strong plasmonic coupling between adjacent gold nanoparticles. It is interesting that secondary assembly of RPA can be triggered by NIR laser irradiation. Prolonged residence time in tumors is achieved after RPA assemblies are fused into superstructures with larger sizes, realizing real‐time monitoring of the therapeutic processes via PAI with enhanced photoacoustic signals. Notably, synergistic effect resulting from PTT‐enhanced chemotherapy is realized to demonstrate high antitumor performance. This work provides a facile strategy to construct flexible photothermal assemblies with favorable properties for imaging‐guided synergistic therapy.     

Precise Deciphering of Brain Vasculatures and Microscopic Tumors with Dual NIR‐II Fluorescence and Photoacoustic Imaging

Diagnostics of cerebrovascular structures and microscopic tumors with intact blood–brain barrier (BBB) significantly contributes to timely treatment of patients bearing neurological diseases. Dual NIR‐II fluorescence and photoacoustic imaging (PAI) is expected to offer powerful strength, including good spatiotemporal resolution, deep penetration, and large signal‐to‐background ratio (SBR) for precise brain diagnostics. Herein, biocompatible and photostable conjugated polymer nanoparticles (CP NPs) are reported for dual‐modality brain imaging in the NIR‐II window. Uniform CP NPs with a size of 50 nm are fabricated from microfluidics devices, which show an emission peak at 1156 nm with a large absorptivity of 35.2 L g^?1 cm^?1 at 1000 nm. The NIR‐II fluorescence imaging resolves hemodynamics and cerebral vasculatures with a spatial resolution of 23 µm at a depth of 600 µm. The NIR‐II PAI enables successful noninvasive mapping of deep microscopic brain tumors (<2 mm at a depth of 2.4 mm beneath dense skull and scalp) with an SBR of 7.2 after focused ultrasound‐induced BBB opening. This study demonstrates that CP NPs are promising contrast agents for brain diagnostics.     

“Dual Lock‐and‐Key”‐Controlled Nanoprobes for Ultrahigh Specific Fluorescence Imaging in the Second Near‐Infrared Window

Fluorescence imaging in the second near‐infrared window (NIR‐II) is a new technique that permits visualization of deep anatomical features with unprecedented spatial resolution. Although attractive, effectively suppressing the interference signal of the background is still an enormous challenge for obtaining target‐specific NIR‐II imaging in the complex and dynamic physiological environment. Herein, dual‐pathological‐parameter cooperatively activatable NIR‐II fluorescence nanoprobes (HISSNPs) are developed whereby hyaluronic acid chains and disulfide bonds act as the “double locks” to lock the fluorescence‐quenched aggregation state of the NIR‐II fluorescence dyes for performing ultrahigh specific imaging of tumors in vivo. The fluorescence can be lit up only when the “double locks” are opened by reacting with the “dual smart keys” (overexpressed hyaluronidase and thiols in tumor) simultaneously. In vivo NIR‐II imaging shows that they reduce nonspecific activitation and achieve ultralow background fluorescence, which is 10.6‐fold lower than single‐parameter activatable probes (HINPs) in the liver at 15 h postinjection. Consequently, these “dual lock‐and‐key”‐controlled HISSNPs exhibit fivefold higher tumor‐to‐normal tissue ratio than “single lock‐and‐key”‐controlled HINPs at 24 h postinjection, attractively realizing ultrahigh specificity of tumor imaging. This is thought to be the first attempt at implementing ultralow background interference with the participation of multiple pathological parameters in NIR‐II fluorescence imaging.     

Molecular Cancer Imaging in the Second Near‐Infrared Window Using a Renal‐Excreted NIR‐II Fluorophore‐Peptide Probe

In vivo molecular imaging of tumors targeting a specific cancer cell marker is a promising strategy for cancer diagnosis and imaging guided surgery and therapy. While targeted imaging often relies on antibody‐modified probes, peptides can afford targeting probes with small sizes, high penetrating ability, and rapid excretion. Recently, in vivo fluorescence imaging in the second near‐infrared window (NIR‐II, 1000–1700 nm) shows promise in reaching sub‐centimeter depth with microscale resolution. Here, a novel peptide (named CP) conjugated NIR‐II fluorescent probe is reported for molecular tumor imaging targeting a tumor stem cell biomarker CD133. The click chemistry derived peptide‐dye (CP‐IRT dye) probe afforded efficient in vivo tumor targeting in mice with a high tumor‐to‐normal tissue signal ratio (T/NT > 8). Importantly, the CP‐IRT probes are rapidly renal excreted (≈87% excretion within 6 h), in stark contrast to accumulation in the liver for typical antibody‐dye probes. Further, with NIR‐II emitting CP‐IRT probes, urethra of mice can be imaged fluorescently for the first time noninvasively through intact tissue. The NIR‐II fluorescent, CD133 targeting imaging probes are potentially useful for human use in the clinic for cancer diagnosis and therapy.     

A Single Composition Architecture‐Based Nanoprobe for Ratiometric Photoacoustic Imaging of Glutathione (GSH) in Living Mice

As one of the reduction species, glutathione (GSH) plays a tremendous role in regulating the homeostasis of redox state in living body. Accurate imaging of GSH in vivo is highly desired to provide a real‐time visualization of physiological and pathological conditions while it is still a big challenge. Recently developed photoacoustic imaging (PAI) with high resolution and deep penetration characteristics is more promising for in vivo GSH detection. However, its application is dramatically limited by the difficult designation of photoacoustic probes with changeable near‐infrared (NIR)‐absorption under reductive activation. A cyanine derivative‐based activatable probe is developed for in vivo ratiometric PAI of GSH for the first time. The probe is structurally designed to output ratiometric signals toward GSH in NIR‐absorption region based on the cleavage of disulfide bond followed by a subsequent exchange between the secondary amine and sulfydryl group formed. Such a ratiometric manner provides high signal‐to‐noise imaging of blood vessels and their surrounding areas in tumor. Concomitantly, it also exhibits good specificity toward GSH over other thiols. Furthermore, the single composition architecture of the probe effectively overcomes the leakage issue compared with traditional multicomposition architecture‐based nanoprobe, thus enhancing the imaging accuracy and fidelity in living body.     

Nanoscale Ultrasound‐Switchable FRET‐Based Liposomes for Near‐Infrared Fluorescence Imaging in Optically Turbid Media

A new approach for fluorescence imaging in optically turbid media centered on the use of nanoscale ultrasound‐switchable FRET‐based liposome contrast agents is reported. Liposomes containing lipophilic carbocyanine dyes as FRET pairs with emission wavelengths located in the near‐infrared window are prepared. The efficacy of FRET and self‐quenching for liposomes with a range of fluorophore concentrations is first calculated from measurement of the liposome emission spectra. Exposure of the liposomes to ultrasound results in changes in the detected fluorescent signal, the nature of which depends on the fluorophores used, detection wavelength, and the fluorophore concentration. Line scanning of a tube containing the contrast agents with 1 mm inner diameter buried at a depth of 1 cm in a heavily scattering tissue phantom demonstrates an improvement in image spatial resolution by a factor of 6.3 as compared with images obtained in the absence of ultrasound. Improvements are also seen in image contrast with the highest obtained being 9% for a liposome system containing FRET pairs. Overall the results obtained provide evidence of the potential the nanoscale ultrasound‐switchable FRET‐based liposomes studied here have for in vivo fluorescence imaging.     

Nanoparticle Regrowth Enhances Photoacoustic Signals of Semiconducting Macromolecular Probe for In Vivo Imaging

Smart molecular probes that emit deep‐tissue penetrating photoacoustic (PA) signals responsive to the target of interest are imperative to understand disease pathology and develop innovative therapeutics. This study reports a self‐assembly approach to develop semiconducting macromolecular activatable probe for in vivo imaging of reactive oxygen species (ROS). This probe comprises a near‐infrared absorbing phthalocyanine core and four poly(ethylene glycol) (PEG) arms linked by ROS‐responsive self‐immolative segments. Such an amphiphilic macromolecular structure allows it to undergo an ROS‐specific cleavage process to release hydrophilic PEG and enhance the hydrophobicity of the nanosystem. Consequently, the residual phthalocyanine component self‐assembles and regrows into large nanoparticles, leading to ROS‐enhanced PA signals. The small size of the intact macromolecular probe is beneficial to penetrate into the tumor tissue of living mice, while the ROS‐activated regrowth of nanoparticles prolongs the retention along with enhanced PA signals, permitting imaging of ROS during chemotherapy. This study thus capitalizes on stimuli‐controlled self‐assembly of macromolecules in conjunction with enhanced heat transfer in large nanoparticles for the development of smart molecular probes for PA imaging.     

Single‐Photomolecular Nanotheranostics for Synergetic Near‐Infrared Fluorescence and Photoacoustic Imaging‐Guided Highly Effective Photothermal Ablation

Multi‐modality imaging‐guided cancer therapy is considered as a powerful theranostic platform enabling simultaneous precise diagnosis and treatment of cancer. However, recently reported multifunctional systems with multiple components and sophisticate structures remain major obstacles for further clinical translation. In this work, a single‐photomolecular theranostic nanoplatform is fabricated via a facile nanoprecipitation strategy. By encapsulating a semiconductor oligomer (IT‐S) into an amphiphilic lipid, water‐dispersible IT‐S nanoparticles (IT‐S NPs) are prepared. The obtained IT‐S NPs have a very simple construction and possess ultra‐stable near‐infrared (NIR) fluorescence (FL)/photoacoustic (PA) dual‐modal imaging and high photothermal conversion efficiency of 72.3%. Accurate spatiotemporal distribution profiles of IT‐S NPs are successfully visualized by NIR FL/PA dual‐modal imaging. With the comprehensive in vivo imaging information provided by IT‐S NPs, tumor photothermal ablation is readily realized under precise manipulation of laser irradiation, which greatly improves the therapeutic efficacy without any obvious side effects. Therefore, the IT‐S NPs allow high tumor therapeutic efficacy under the precise guidance of FL/PA imaging techniques and thus hold great potential as an effective theranostic platform for future clinical applications.     

Plasmonic enhancement of fluorescence on silver nanoparticle films

The localized plasmon controlled fluorescence has been discussed by comparing the fluorescence enhancement of dyes on different shaped silver nanoparticle self-assembled films. A trilayer structure, composed of a silver nanoparticle monolayer, a proper thickness polyelectrolyte spacing layer and a dye-adsorbed layer, was constructed to study the plasmon enhanced fluorescence properties. The effective coupling of the plasmon band with the excitation or emission of dye resulted in different enhancement factors. Moreover, the plasmon enhanced fluorescence resonance energy transfer (FRET) of two dyes was observed. The FRET efficiency of the spherical silver nanoparticle self-assembled film had a 2.8-fold increase. The improvement of FRET efficiency via localized surface plasmons would increase the sensitivity of FRET-based bioassays.     

pH-stimuli-responsive near-infrared optical imaging nanoprobe based on poly(γ-glutamic acid)/poly(β-amino ester) nanoparticles

pH-stimuli-responsive near-infrared optical imaging nanoprobes are designed and synthesized in this study in a facile one-step synthesis process based on the use of the biocompatible and biodegradable polymer poly(γ-glutamic acid) (γ-PGA)/poly(β-amino ester) (PBAE). PBAE has good transfection efficiency and promotes degradation properties under acidic conditions. This pH-responsive degradability can be used for the effective release of encapsulating materials after cellular uptake. As an optical imaging probe, indocyanine green (ICG) is an FDA-approved near-infrared fluorescent dye with a quenching property at a high concentration. In this regard, we focus here on the rapid degradation of PBAE in an acidic environment, in which the nanoparticles are disassembled. This allows the ICG dyes to show enhanced fluorescence signals after being releasing from the particles. We demonstrated this principle in cellular uptake experiments. We expect that the developed pH-stimuli-responsive smart nanoprobes can be applied in intracellular delivery signaling applications.     

Activatable Semiconducting Theranostics: Simultaneous Generation and Ratiometric Photoacoustic Imaging of Reactive Oxygen Species In Vivo

Enhancing the generation of reactive oxygen species (ROS) is an effective anticancer strategy. However, it is a great challenge to control the production and to image ROS in vivo, both of which are vital for improving the efficacy and accuracy of cancer therapy. Herein, an activatable semiconducting theranostic nanoparticle (NP) platform is developed that can simultaneously enhance ROS generation while self‐monitoring its levels through ratiometric photoacoustic (PA) imaging. The NP platform can further guide in vivo therapeutic effect in tumors. The theranostic NP platform is composed of: (i) cisplatin prodrug and ferric ion catalyst for ROS generation, a part of combination cancer therapy; and (ii) a ratiometric PA imaging nanoprobe consisting of inert semiconducting perylene‐diimide (PDI) and ROS activatable near‐infrared dye (IR790s), used in ratiometric PA imaging of ROS during cancer treatment. Ratiometric PA signals are measured at two near‐infrared excitation wavelengths: 680 and 790 nm for PDI and IR790s, respectively. The measurements show highly accurate visualization of ^?OH generation in vivo. This novel ROS responsive organic theranostic NP allows not only synergistic cancer chemotherapy but also real‐time monitoring of the therapeutic effect through ratiometric PA imaging.     

Guiding Brain‐Tumor Surgery via Blood–Brain‐Barrier‐Permeable Gold Nanoprobes with Acid‐Triggered MRI/SERRS Signals

Surgical resection is a mainstay in the treatment of malignant brain tumors. Surgeons, however, face great challenges in distinguishing tumor margins due to their infiltrated nature. Here, a pair of gold nanoprobes that enter a brain tumor by crossing the blood–brain barrier is developed. The acidic tumor environment triggers their assembly with the concomitant activation of both magnetic resonance (MR) and surface‐enhanced resonance Raman spectroscopy (SERRS) signals. While the bulky aggregates continuously trap into the tumor interstitium, the intact nanoprobes in normal brain tissue can be transported back into the blood stream in a timely manner. Experimental results show that physiological acidity triggers nanoparticle assembly by forming 3D spherical nanoclusters with remarkable MR and SERRS signal enhancements. The nanoprobes not only preoperatively define orthotopic glioblastoma xenografts by magnetic resonance imaging (MRI) with high sensitivity and durability in vivo, but also intraoperatively guide tumor excision with the assistance of a handheld Raman scanner. Microscopy studies verify the precisely demarcated tumor margin marked by the assembled nanoprobes. Taking advantage of the nanoprobes' rapid excretion rate and the extracellular acidification as a hallmark of solid tumors, these nanoprobes are promising in improving brain‐tumor surgical outcome with high specificity, safety, and universality.     

Metabolizable Semiconducting Polymer Nanoparticles for Second Near‐Infrared Photoacoustic Imaging

Photoacoustic (PA) imaging in the second near‐infrared (NIR‐II) window (1000–1700 nm) holds great promise for deep‐tissue diagnosis due to the reduced light scattering and minimized tissue absorption; however, exploration of such a noninvasive imaging technique is greatly constrained by the lack of biodegradable NIR‐II absorbing agents. Herein, the first series of metabolizable NIR‐II PA agents are reported based on semiconducting polymer nanoparticles (SPNs). Such completely organic nanoagents consist of π‐conjugated yet oxidizable optical polymer as PA generator and hydrolyzable amphiphilic polymer as particle matrix to provide water solubility. The obtained SPNs are readily degraded by myeloperoxidase and lipase abundant in phagocytes, transforming from nonfluorescent nanoparticles (30 nm) into NIR fluorescent ultrasmall metabolites (≈1 nm). As such, these nanoagents can be effectively cleared out via both hepatobiliary and renal excretions after systematic administration, leaving no toxicity to living mice. Particularly these nanoagents possess high photothermal conversion efficiencies and emit bright PA signals at 1064 nm, enabling sensitive NIR‐II PA imaging of both subcutaneous tumor and deep brain vasculature through intact skull in living animals at a low systematic dosage. This study thus provides a generalized molecular design toward organic metabolizable semiconducting materials for biophotonic applications in NIR‐II window.     

Miniature Hollow Gold Nanorods with Enhanced Effect for In Vivo Photoacoustic Imaging in the NIR‐II Window

The miniaturization of gold nanorods exhibits a bright prospect for intravital photoacoustic imaging (PAI) and the hollow structure possesses a better plasmonic property. Herein, miniature hollow gold nanorods (M‐AuHNRs) (≈46 nm in length) possessing strong plasmonic absorbance in the second near‐infrared (NIR‐II) window (1000–1350 nm) are developed, which are considered as the most suitable range for the intravital PAI. The as‐prepared M‐AuHNRs exhibit 3.5 times stronger photoacoustic signal intensity than the large hollow Au nanorods (≈105 nm in length) at 0.2 optical density under 1064 nm laser irradiation. The in vivo biodistribution measurement shows that the accumulation in tumor of miniature nanorods is twofold as high as that of the large counterpart. After modifying with a tumor‐targeting molecule and fluorochrome, in living tumor‐bearing mice, the M‐AuHNRs group gives a high fluorescence intensity in tumors, which is 3.6‐fold that of the large ones with the same functionalization. Moreover, in the intravital PAI of living tumor‐bearing mice, the M‐AuHNRs generate longer‐lasting and stronger photoacoustic signal than the large counterpart in the NIR‐II window. Overall, this study presents the fabrication of M‐AuHNRs as a promising contrast agent for intravital PAI.     

Liposomal Encapsulation of a Near‐Infrared Fluorophore Enhances Fluorescence Quenching and Reliable Whole Body Optical Imaging Upon Activation In Vivo

In the past decade, there has been significant progress in the development of water soluble near‐infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650–900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto‐fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY‐676‐COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY‐676‐COOH reveal strong fluorescence quenching. It is demonstrated that the non‐targeted PEGylated fluorescence‐activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan‐induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY‐676‐COOH, prolonged stability and retention of liposomal‐DY‐676‐COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY‐676‐COOH‐loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases.     

Near‐Infrared Fluorescent Ag2Se–Cetuximab Nanoprobes for Targeted Imaging and Therapy of Cancer

Theranostic nanoprobes integrated with diagnostic imaging and therapy capabilities have shown great potential for highly effective tumor therapy by realizing imaging‐guided drug delivery and tumor treatment. Developing novel high‐performance nanoprobes is an important basis for tumor theranostic application. Here, near‐infrared (NIR) fluorescent and low‐biotoxicity Ag₂Se quantum dots (QDs) have been coupled with cetuximab, a clinical antiepidermal growth factor receptor antibody drug for tumor therapy, via a facile bioconjugation strategy to prepare multifunctional Ag₂Se–cetuximab nanoprobes. Compared with the Ag₂Se QDs alone, the Ag₂Se–cetuximab nanoprobes display faster and more enrichment at the site of orthotopic tongue cancer, and thus present better NIR fluorescence contrast between the tumor and the surrounding regions. At 24 h postinjection, the NIR fluorescence of Ag₂Se–cetuximab nanoprobes at the tumor site is still easily detectable, whereas no fluorescence is observed for the Ag₂Se QDs. Moreover, the Ag₂Se–cetuximab nanoprobes have also significantly inhibited the tumor growth and improved the survival rate of orthotopic tongue cancer‐bearing nude mice from 0% to 57.1%. Taken together, the constructed multifunctional Ag₂Se–cetuximab nanoprobes have achieved combined targeted imaging and therapy of orthotopic tongue cancer, which may greatly contribute to the development of nanotheranostics.