Near-infrared Fluorescence Signal Research Articles

Fabrication of a Near-Infrared-Emissive Probe for Detecting Dipeptidyl Peptidase 4 in the Liver of Diabetic Mice and Clinical Serum.

Dipeptidyl peptidase 4 (DPP4) plays a key role in glucose metabolism, which has been a close target for diabetes pathology and treatment. It is significant for the evaluation of cellular DPP4 activity in various biological systems. Fluorescence imaging technology is currently a popular method for detecting enzymes in living cells due to its advantages of high selectivity, high sensitivity, high spatiotemporal resolution, and real-time visualization. Herein, a near-infrared (NIR)-emissive probe NEDP with a large Stokes shift (153 nm) was developed for the assay of DPP4 activity. Upon addition of DPP4, NEDP can emit a significant turn-on NIR fluorescence signal (673 nm) with high sensitivity and specificity. Moreover, NEDP can successfully be used for imaging of intracellular DPP4, confirming the regulation of DPP4 expression in hyperglucose and its treatment in living cells. Most importantly, NEDP can not only monitor the changes of DPP4 in vivo but also show that DPP4 in diabetes is mainly up-regulated in the liver, and the level of DPP4 is positively correlated with the pathological damage of the liver. In addition, NEDP can identify the serum of diabetic patients from healthy people through the fluorescence response to DPP4. These results demonstrated that the designed probe NEDP provides a prospective visual tool to explore the relationship between DPP4 and diabetes and would be applied for detecting serum of diabetes in the clinic.

Guest-Responsive Near-Infrared-Luminescent Metal-Organic Cage Organized by Porphyrin Dyes and Yb(III) Complexes.

Metal-organic cages (MOCs) with luminophores have significant advantages for the facile detection of specific molecules based on turn-on or turn-off luminescence changes induced by host-guest complexation. One important challenge is the development of turn-on-type near-infrared (NIR)-luminescent MOCs. In this study, we synthesized a novel MOC consisting of two porphyrin dyes linked by four Yb(III) complexes, which exhibit bimodal red and NIR fluorescence signals upon photoexcitation of the porphyrin π system. Single-crystal X-ray structural analysis and computational molecular modeling revealed that planar aromatic perfluorocarbons were intercalated into the MOC. The tight packing between the MOC and guests enhanced the NIR fluorescence of Yb(III) by suppressing energy transfer from the photoexcited porphyrin to oxygen molecules. Guest-responsive turn-on NIR fluorescence changes in an MOC were successfully demonstrated.

A General Cyanine‐Based Platform for Designing Robust Dual‐Channel Near‐Infrared Fluorescent and Photoacoustic Probes

AbstractThe creation of versatile platforms for developing dual‐channel near‐infrared fluorescent (NIRF) and photoacoustic (PA) probes, especially those engineered to minimize channel crosstalk, is crucial for precise biomarker detection. However, such platforms remain scarce. To bridge this gap, this study introduces an innovative cyanine‐based platform, CySN. The CySN platform showcases remarkable wavelength‐shifting properties, including large fluorescent modality shift (68 nm) and PA modality shift (145 nm) after the decaging reaction. These substantial changes lead to an exceptionally high ratiometric NIRF change of 603‐fold and ratiometric PA change of 261‐fold. Leveraging the CySN platform, dual‐channel NIRF/PA probes have been successfully developed for detecting both small molecule biomarker (H2O2) and enzyme biomarker (esterase). These probes demonstrate the ability to detect their targets through dual‐channel NIRF/PA detection with high sensitivity and selectivity in vitro. Furthermore, the probes effectively harness NIRF signals to image target analytes in living cells. Notably, the probes demonstrate the capability to accurately diagnose tumors by detecting tumor markers (H2O2 and esterase), revealing a 3.6 to 7‐fold ratiometric PA enhancement over normal tissue. Therefore, the CySN platform holds the potential to further advance the development of dual‐channel NIRF/PA probes for biomolecule detection in disease diagnosis.

NIR-activated water-soluble molecular probe based on Cu(ii) ion quenching mechanisms and substituted strategies: For in vitro/in vivo H2S detection

Human health, environmental contamination, and food safety are tightly associated with hydrogen sulfide (H2S). The safe and highly sensitive monitoring of H2S in tissues, organs, and blood has presented ongoing challenges. Herein, a novel near-infrared (NIR)-activated water-soluble molecular probe, denoted as IR820-TPY-Cu was developed for detecting hydrogen sulfide. This probe is built upon the fluorescent molecule IR820, a derivative of Indocyanine green (ICG). Upon reacting with H2S, IR820-TPY-Cu exhibits active fluorescence (670–750 nm), which is attributed to the Cu (II) ion quenching mechanism and substitution strategy. IR820-TPY-Cu exhibits selectively activated near-infrared fluorescence signal changes, a low detection limit of 0.114 μM, and a rapid in vitro response (3 min) for H2S detection. Moreover, the biocompatible probe IR820-TPY-Cu not only quantitatively monitors H2S in environmental water and various food specimens (red wine, beer, meat, milk, and sweet potato) through colorimetric sensing but also successfully images endogenous and exogenous H2S in living cells, zebrafish, and mice. This research provides a promising approach for monitoring H2S in environmental contamination, ensuring food safety, and studying living systems.

Albumin-mediated molecular competition of supramolecular photosensitizers for NIR-II imaging-guided phototherapy

Heptamethine cyanines (Cy7) typically exhibit strong near-infrared (NIR) fluorescence signals when in monodisperse states, but their distinctive π-conjugated structure leads to limited solubility in aqueous solutions, ultimately resulting in inherent fluorescence self-quenching. Enhancing the fluorescence quantum yield of these agents within aqueous environments, in order to achieve high-resolution biological imaging, presents a significant challenge. Herein, a benzothiazole-based Cy7-derivative (Cy7-BTH) is developed, exhibiting unique H-aggregation properties in aqueous solutions. Bovine serum albumin (BSA) binds to Cy7-BTH (BSA@Cy7-BTH) to modulate its molecular stacking and energy transfer, boosting its fluorescence accompanied by bright NIR-II tail emission. The competitive binding alters the planarity of Cy7-BTH, restricting its intramolecular rotation, and simutaneously changes the conformation of BSA, leading to BSA@Cy7-BTH aggregation to form hierarchical-structured nanoparticles. Notably, the BSA@Cy7-BTH nanoparticles significantly enhanced photothermal response of Cy7-BTH while maintaining its controlled reactive oxygen species (ROS) generation, offering NIR-II imaging-guided surgical removal of primary tumors, and simultaneously inhibiting cancer cell metastasis by synergistic PDT/PTT dissection of metastatic tumor cells in sentinel lymph nodes (SLNs). This study provides an innovative but facile strategy for their potential clinical applications by precisely regulating the intermolecular competitive interactions of NIR theranostic agents.

A β-Galactosidase-Activated Fluorogenic Reporter for the Detection of Gastric Cancer In Vivo and in Urine.

Although gastric cancer (GC) is one of the most frequent malignant tumors in the digestive tract with high morbidity and mortality, it remains a diagnostic dilemma due to its reliance on invasive biopsy or insensitive assays. Herein, we report a fluorescent gastric cancer reporter (FGCR) with activatable near-infrared fluorescence (NIRF) signals and high renal-clearance efficiency for the detection of orthotopic GC in a murine model via real-time imaging and remote urinalysis. In the presence of gastric-tumor-associated β-galactosidase (β-Gal), FGCR can be fluorescently activated for in vivo NIRF imaging. Relying on its high renal-clearance efficiency (∼95% ID), it can be rapidly excreted through kidneys to urine for the ultrasensitive detection of tumors with a diameter down to ∼2.1 mm and for assessing the prognosis of oxaliplatin-based chemotherapy. This study not only provides a new approach for noninvasive auxiliary diagnosis and prognosis of GC but also provides guidelines for the development of fluorescence probes for cancer diagnosis.

Targeted Biodegradable Near-Infrared Fluorescent Nanoparticles for Colorectal Cancer Imaging.

Colorectal cancer (CRC) is the third leading cause of cancer death in the U.S., and early detection and diagnosis are essential for effective treatment. Current methods are inadequate for rapid detection of early disease, revealing flat lesions, and delineating tumor margins with accuracy and molecular specificity. Fluorescence endoscopy can generate wide field-of-view images enabling detection of CRC lesions and margins; increased signal intensity and improved signal-to-noise ratios can increase both speed and sensitivity of cancer detection. For this purpose, we developed targeted near-infrared (NIR) fluorescent silica nanoparticles (FSNs). We tuned their size to 50-200 nm and conjugated their surface with an antibody to carcinoembryonic antigen (CEA) to prepare CEA-FSNs. The physicochemical properties and biodegradable profiles of CEA-FSN were characterized, and molecular targeting was verified in culture using HT29 (CEA positive) and HCT116 (CEA negative) cells. CEA-FSNs bound to the HT29 cells to a greater extent than to the HCT116 cells, and smaller CEA-FSNs were internalized into HT29 cells more efficiently than larger CEA-FSNs. After intravenous administration of CEA-FSNs, a significantly greater signal was observed from the CEA-positive HT29 than the CEA-negative HCT116 tumors in xenografted mice. In F344-PIRC rats, polyps in the intestine were detected by white-light endoscopy, and NIR fluorescent signals were found in the excised intestinal tissue after topical application of CEA-FSNs. Immunofluorescence imaging of excised tissue sections demonstrated that the particle signals coregistered with signals for both CRC and CEA. These results indicate that CEA-FSNs have potential as a molecular imaging marker for early diagnosis of CRC.

Rational design of a near-infrared fluorescent probe for monitoring butyrylcholinesterase activity and its application in development of inhibitors.

Butyrylcholinesterase (BChE) is widely expressed in multiple tissues and has a vital role in several key human disorders, such as Alzheimer's disease and tumorigenesis. However, the role of BChE in human disorders has not been investigated. Thus, to quantitatively detect and visualize dynamical variations in BChE activity is essential for exploring the biological roles of BChE in the progression of a number of human disorders. Herein, based on the substrate characteristics of BChE, we customized and synthesized three near-infrared (NIR) fluorescent probe substrates with cyanine-skeleton, and finally selected a NIR fluorescence probe substrate named CYBA. The CYBA demonstrated a significant increase in fluorescence when interacting with BChE, but mainly avoided AChE. Upon the addition of BChE, CYBA could be specifically hydrolyzed to TBO, resulting in a significant NIR fluorescence signal enhancement at 710nm. Systematic evaluation revealed that CYBA exhibited exceptional chemical stability in complex biosamples and possessed remarkable selectivity and sensitivity towards BChE. Moreover, CYBA was successfully applied for real-time imaging of endogenous BChE activity in two types of nerve-related living cells. Additionally, CYBA demonstrated exceptional stability in the detection of complex biological samples in plasma recovery studies (97.51%-104.01%). Furthermore, CYBA was used to construct a high-throughput screening (HTS) method for BChE inhibitors using human plasma as the enzyme source. We evaluated inhibitory effects of a series of natural products and four flavonoids were identified as potent inhibitors of BChE. Collectively, CYBA can serve as a practical tool to track the changes of BChE activity in complicated biological environments due to its excellent capabilities.

Using ICG navigation in resection of a horseshoe kidney

BACKGROUND: In this publication, we present a new area of application of indocyanine green (ICG) imaging in pediatric surgery. We used this advanced method to evaluate the resection margins of a functional segment of the horseshoe kidney after intravenous indocyanine green. CLINICAL CASE DESCRIPTION: The article presents a case of treatment of a horseshoe kidney complicated by vesicorenal reflux in the left segment and loss of function of the left segment. Resection of the left half of the horseshoe kidney was performed using fluoroscopic control with intravenous administration of indocyanine green. The surgery was performed using the RUBINA™ endovideosurgical system manufactured by KARL STORZ (Germany). RUBINA™ components offer a variety of ICG in near-infrared fluorescence signal imaging modes. Depending on the surgeon and the application, ICG in near-infrared fluorescence data can be used in different modes: image overlay mode, backup mode, color mapping mode. After confirming the boundaries of the perfused part of the kidney, laparoscopic resection of the functional half was performed at the level of the isthmus. The use of ICG imaging was very important in identifying healthy tissue that stained intensely green 2 minutes after intravenous indocyanine green administration. This margin check verifies the functioning renal parenchyma and allows the correct selection of the nephrectomy margin. The next day after the operation, an ultrasound examination of the renal remnant and perinephric space was performed — the rotation to the right hour of the horseshoe-shaped kidney was good and no accumulation of fluid was detected in the bed of the removed segment. A control ultrasound examination 6 months after the operation showed the preservation of the parenchyma of the right half of the kidney, which indicates the positive effect of the surgical procedure. CONCLUSION: ICG navigation is a promising method for noninvasive assessment of resection margins in patients with horseshoe kidneys.

β-Galactosidase-Activated and Red Light-Induced RNA Modification Strategy for Prolonged NIR Fluorescence/PET Bimodality Imaging.

Improving the retention of small-molecule-based therapeutic agents in tumors is crucial to achieve precise diagnosis and effective therapy of cancer. Herein, we propose a β-galactosidase (β-Gal)-activated and red light-induced RNA modification (GALIRM) strategy for prolonged tumor imaging. A β-Gal-activatable near-infrared (NIR) fluorescence (FL) and positron emission tomography (PET) bimodal probe 68Ga-NOTA-FCG consists of a triaaza triacetic acid chelator NOTA for 68Ga-labeling, a β-Gal-activated photosensitizer CyGal, and a singlet oxygen (1O2)-susceptible furan group for RNA modification. Studies have demonstrated that the probe emits an activated NIR FL signal upon cleavage by endogenous β-Gal overexpressed in the lysosomes, which is combined with the PET imaging signal of 68Ga allowing for highly sensitive imaging of ovarian cancer. Moreover, the capability of 68Ga-NOTA-FCG generating 1O2 under 690 nm illumination could be simultaneously unlocked, which can trigger the covalent cross-linking between furan and nucleotides of cytoplasmic RNAs. The formation of the probe-RNA conjugate can effectively prevent exocytosis and prolong retention of the probe in tumors. We thus believe that this GALIRM strategy may provide entirely new insights into long-term tumor imaging and efficient tumor treatment.

Preparation of near-infrared photoacoustic imaging and photothermal treatment agent for cancer using a modifiable acid-triggered molecular platform.

Ratiometric near-infrared fluorescent pH probes with various pKa values were innovatively designed and synthesized based on cyanine with a diamine moiety. The photochemical properties of these probes were thoroughly evaluated. Among the series, IR-PHA exhibited an optimal pKa value of approximately 6.40, closely matching the pH of cancerous tissues. This feature is particularly valuable for real-time pH monitoring in both living cells and living mice. Moreover, when administered intravenously to tumor-bearing mice, IR-PHA demonstrated rapid and significant enhancement of near-infrared fluorescence and photoacoustic signals within the tumor region. This outcome underscores the probe's exceptional capability for dual-modal cancer imaging utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) modalities. Concurrently, the application of a continuous-wave near-infrared laser efficiently ablated cancer cells in vivo, attributed to the photothermal effect induced by IR-PHA. The results strongly indicate that IR-PHA is well-suited for NIRF/PA dual-modality imaging and photothermal therapy of tumors. This makes it a promising candidate for theranostic applications involving small molecules.

Construction of a novel aminofluorene-based ratiometric near-infrared fluorescence probe for detecting carboxylesterase activity in living cells.

Herein, we constructed a novel aminofluorene-based fluorescence probe (FEN-CE) for the detection of carboxylesterase (CE) in living cells by a ratiometric near-infrared (NIR) fluorescence signal. FEN-CE with NIR emission (650 nm) could be hydrolyzed specifically by CE and transformed to FENH with the release of the self-immolative group, which exhibited a red-shifted emission peak of 680 nm. In addition, FEN-CE showed high selectivity for CE and was successfully used in the detection of CE activity in living cells through its ratiometric NIR fluorescence signals.

A near-infrared fluorescent probe with a substantial Stokes shift designed for the detection and imaging of β-galactosidase within living cells and animals

β-Galactosidase serves as a pivotal biomarker for both cancer and cellular aging. The advancement of fluorescent sensors for tracking β-galactosidase activity is imperative in the realm of cancer diagnosis. We have designed a near-infrared fluorescent probe (PTA-gal) for the detection of β-galactosidase in living systems with large Stokes shifts. PTA-gal exhibits remarkable sensitivity and selectivity in detecting β-galactosidase, producing near-infrared fluorescent signals with a remarkably low detection limit (2.2 × 10-5 U/mL) and a high quantum yield (0.30). Moreover, PTA-gal demonstrates biocompatibility and can effectively detect β-galactosidase in cancer cells as well as within living animals.

Engineering a “turn-on” NIR fluorescent sensor-based hydroxyphenyl benzothiazole with a cinnamoyl unit for hydrazine and its environmental and in-vitro applications

Hydrazine (N2H4), a chemical compound widely used in various industrial applications, causes significant environmental and biological hazards. Therefore, it is crucial to develop methodologies for the visualization and real time tracking of N2H4. In this regard, we have constructed a novel near-infrared fluorescent probe (HBT-Cy) that can effectively detect N2H4 in various samples. HBT-Cy contains 2-(2′-hydroxyphenyl)benzothiazole (HBT), cinnamoyl (Cy), and pyridinium (Py) moieties. Importantly, HBT-Cy exhibits a rapid, selective, and highly sensitive response to N2H4. This response results in the release of HBT-Py and the generation of considerable colorimetric changes along with a significant NIR (near infrared) fluorescence signal, peaking at 685 nm. Advantages of this system include turn on NIR fluorescence with large Stokes shift, (approximately 171 nm), low limit of detection (LOD = 0.11 μM) and quantum yield (0.211). The probe with low cytotoxic behavior demonstrates strong NIR fluorescence imaging capabilities to visualize endogenous and exogenous N2H4 in live cells. This mitochondria-targetable probe shows effective subcellular localization. These results suggest that HBT-Cy is a valuable probe for tracking and investigating the behavior of N2H4 in biological systems and environmental samples.

Tailored Zwitterionic Hemicyanine Reporters for Early Diagnosis and Prognostic Assessment of Acute Renal Failure.

Drug-induced renal failure (DIRF) poses a serious medical complication with high mortality risk. However, early diagnosis or prognosis of DIRF remain challenging, as current methods rely on detecting late-stage biomarkers. Herein we present a library of zwitterionic unimolecular hemicyanines (ZCs) available for constructing activatable reporters to detect DIRF since its initial stage. Zwitterionic properties of these probes are achieved through interspersedly integrating alkyl sulfonates and quaternary ammonium cations onto hemicyanine skeleton, which result in record low plasma protein binding (<5 %) and remarkable renal clearance efficiencies (≈96 %). An activatable reporter ZCRR is further developed by masking the optimal candidate ZC6 with a tetrapeptide specifically cleavable by caspase-8, an initiating indicator of apoptosis. In living mice with cisplatin-induced DIRF, systematically administered ZCRR efficiently accumulates in kidneys and responds to elevated caspase-8 for near-infrared fluorescence signals 'turn-on', enabling sensitive detection of intrarenal apoptosis 60 h earlier than clinical methods, and precise evaluation of apoptosis remediation effects by different medications on DIRF mice. As it's urinary excretable, ZCRR also allows for remote detection of DIRF and predicting renoprotective efficacy through in vitro optical urinalysis. This study thus presents unimolecular renal clearable scaffolds that are applicable to developing versatile activatable reporters for renal diseases management.

Coordinated modulation of long non-coding RNA ASBEL and curcumin co-delivery through multicomponent nanocomplexes for synchronous triple-negative breast cancer theranostics

BackgroundAbnormally regulated long non-coding RNAs (lncRNAs) functions in cancer emphasize their potential to serve as potential targets for cancer therapeutic intervention. LncRNA ASBEL has been identified as oncogene and an anti-sense transcript of tumor-suppressor gene of BTG3 in triple-negative breast cancer (TNBC).ResultsHerein, multicomponent self-assembled polyelectrolyte nanocomplexes (CANPs) based on the polyelectrolytes of bioactive hyaluronic acid (HA) and chitosan hydrochloride (CS) were designed and prepared for the collaborative modulation of oncogenic lncRNA ASBEL with antago3, an oligonucleotide antagonist targeting lncRNA ASBEL and hydrophobic curcumin (Cur) co-delivery for synergetic TNBC therapy. Antago3 and Cur co-incorporated CANPs were achieved via a one-step assembling strategy with the cooperation of noncovalent electrostatic interactions, hydrogen-bonding, and hydrophobic interactions. Moreover, the multicomponent assembled CANPs were ulteriorly decorated with a near-infrared fluorescence (NIRF) Cy-5.5 dye (FCANPs) for synchronous NIRF imaging and therapy monitoring performance. Resultantly, MDA-MB-231 cells proliferation, migration, and invasion were efficiently inhibited, and the highest apoptosis ratio was induced by FCANPs with coordination patterns. At the molecular level, effective regulation of lncRNA ASBEL/BTG3 and synchronous regulation of Bcl-2 and c-Met pathways could be observed.ConclusionAs expected, systemic administration of FCANPs resulted in targeted and preferential accumulation of near-infrared fluorescence signal and Cur in the tumor tissue. More attractively, systemic FCANPs-mediated collaborative modulating lncRNA ASBEL/BTG3 and Cur co-delivery significantly suppressed the MDA-MB-231 xenograft tumor growth, inhibited metastasis and extended survival rate with negligible systemic toxicity. Our present study represented an effective approach to developing a promising theranostic platform for combating TNBC in a combined therapy pattern.

Specific imaging of intracellular hydrogen sulfide by a positively charged NIR fluorescent probe

The poor water solubility of traditional activatable organic molecular probes usually limits their detection ability in physiological environment. In this work, a positively charged H2S probe was designed, which exhibited a significantly enhanced responsiveness to H2S in the aggregated state due to the increased positive charge density on the aggregate surface. Under physiological conditions, the probe could be activated by H2S with specificity and sensitivity to release near-infrared fluorescence signal. Moreover, endogenous H2S levels in living cells were successfully monitored by using this probe. We expect that this probe can provide a new strategy for the design of activatable probes to break the limitation of poor water solubility of conventional organic molecular probes.

A Dicyanoisoflurone-based Near-infrared Fluorescence Probe for Highly Sensitive Detection of Hg2.

Due to its high toxicity, long durability, easy absorption by aquatic organisms, and significant bioaccumulation, Hg2+ has caused substantial environmental damage and posed serious threats to human health. Therefore, effective detection of Hg2+ is of utmost importance. In this study, a turn-on fluorescent probe based on dicyanoisoflurone was developed for the detection of Hg2+. The probe exhibited near-infrared fluorescence signal at 660nm upon excitation by 440nm UV light in a mixture of CH3CN and HEPES buffer (4:1, v/v, 10 mM, pH = 7.5), with selective binding to Hg2+ in a molar ratio of 1:1. This binding event was accompanied by a visible color change from light yellow to orange. By utilizing the enhanced fluorescence signal change, this probe enables highly sensitive analysis and detection of Hg2+ with excellent selectivity (association constant = 1.63 × 104M- 1), large Stokes shift (220nm), high sensitivity (detection limit as low as 5.6 nM), short reaction time (30s), and a physiological pH range of 7.5-9.5. The probe was successfully employed for detecting of Hg2+ in real water and living cells.

In Situ imaging of orthotopic kidney cancer in the murine model using an aminopeptidase N activatable fluorescent probe

Surgical treatment is the most effective treatment for renal cancer. It is urgent to develop highly selective and sensitive analytical methods for specially lighting up the kidney tumor, so as to achieve accurate imaging-guided surgical resection of cancer tissue. However, the main challenge in real-time and in situ fluorescence imaging of kidney tumor rest with the design of fluorescent probes that not only show renal metabolic pathway to enrich in the kidney cancer cells but also have high activatable signal towards kidney tumor-associated biomarkers. Since aminopeptidase N (APN) acted as a kidney cancer-specific biomarker, herein, a low MW PEGylation and APN-activatable fluorescent probe (HDAPN-PEG) was constructed for in situ near infrared fluorescence (NIRF) visualizing the orthotopic kidney cancer in living mice. HDAPN-PEG was specially activated by APN with good sensitivity and selectivity. HDAPN-PEG dramatically turned on its NIRF signal in kidney cancer cells but kept silent in kidney normal cells. Because of the high renal enrichment and its cancer biomarker specificity, HDAPN-PEG can light up the kidney cancer for in situ imaging of kidney cancer in living mice. This work thus recommends an activable molecular probe that not only promotes mechanistic investigation on the roles of APN in kidney cancer, but also facilitates in situ detection kidney cancer and fluorescence imaging guided nephron sparing surgery.

Artificial Urinary Biomarkers for Early Diagnosis of Acute Renal Allograft Rejection.

Acute renal allograft rejection (ARAR) after kidney transplantation associated with reduced graft survival and eventual graft failure is poorly diagnosed in hospitals. Here, we report the development of artificial biomarker probes (AMPros) for sensitive urinalysis of ARAR in murine models. AMPros spontaneously go to the kidneys after systemic administration, specifically react with the prodromal immune biomarkers to activate their near-infrared fluorescence signals to report cell-mediated rejection, and efficiently undergo renal excretion into urine. Thus, AMPros enable convenient optical urinalysis that detects ARAR prior to histological manifestation of rejection, which is also earlier than current diagnostic methods measuring proinflammatory cytokines and peripheral blood lymphocyte mRNAs. Due to the high kidney specificity, AMPros-based urinalysis discriminates allograft rejection against other non-alloimmune specific diseases, which is unattainable by measurement of serological biomarkers. Such a noninvasive and sensitive urine test holds great promise in continuous monitoring of renal allograft conditions at low resource settings for timely clinical interventions.