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Published in final edited form as: Curr Opin Chem Biol. 2023 Apr 27;74:102310. doi: 10.1016/j.cbpa.2023.102310

Activity-based bioluminescence probes for in vivo sensing applications

Anuj K Yadav 1, Jefferson Chan 1
PMCID: PMC10225331  NIHMSID: NIHMS1889043  PMID: 37119771

Abstract

Bioluminescence imaging is a highly sensitive technique commonly used for various in vivo applications. Recent efforts to expand the utility of this modality has led to the development of a suite of activity-based sensing (ABS) probes for bioluminescence imaging by ‘caging’ of luciferin and its structural analogs. The ability to selectively detect a given biomarker has presented researchers with many exciting opportunities to study both health and disease states in animal models. Here, we highlight recent (2021–2023) bioluminescence-based ABS probes with an emphasis of probe design and in vivo validation experiments.

1. Introduction

Organisms such as the terrestrial firefly, some marine organisms, and glowing fungi have intrigued humans for centuries owing to their remarkable ability to produce light in a process known as bioluminescence [1]. In general, substrates such as luciferin are transformed by specialized enzymes known as luciferases to yield high energy intermediates, which upon collapse, releases a photon of light. Because bioluminescence does not require an external light source, it avoids the consequences of autofluorescence, photobleaching, and phototoxicity to provide exquisite sensitivity (high signal-to-noise ratios) [2,3]. These properties have since been exploited to visualize protein-protein interactions, the manipulation of genes, and the activity of enzymes in luciferase expressing cells and animal models [4–6]. It has also found wide applications in studying various types of diseases such as cancer [7] and infections [8], as well as how the body response during injury [9].

In order to detect a biomarker associated with these conditions via bioluminescence, the field has turned to activity-based sensing (ABS), where a luciferase substrate is ‘caged’ with a biomarker-responsive trigger. Prior to activation, an ABS probe cannot engage with luciferase, and therefore no bioluminescence is generated. However, uncaging of the trigger results in release of the substrate which can then react with luciferase to generate light [10*,11]. The most common site for trigger installation is at the phenolic alcohol of luciferin or in the case of aminoluciferin, the aniline can be modified [10*]. Alternatively, triggers can also be appended to the terminal carboxylate group. Advances in this area have led to the development of ABS probes to visualize the activity of various enzymatic systems (e.g., caspases) [12,13], small molecule analytes (e.g., H2S) [14], and metal ions (e.g., copper) [15–17**]. Of note, ABS designs are unique in the sense that the reactivity of a trigger can be tuned for a given application. For instance, in the event a biomarker is fleeting, triggers with heightened reactivity can be employed. Likewise, if a biomarker is abundant, a trigger can be modified to attenuate reactivity. However, it is notable that in order to report on the target, the reaction kinetics for this first step must be rate-limiting (i.e., faster than the reaction between the liberated substrate and luciferase) [18].

In this article we will highlight select studies published within the last two years featuring ABS probes for in vivo applications. In the first section, we will focus on probe designs based on the native luciferin/luciferase pair. This will be followed by recent examples utilizing near infrared (NIR) emitting luciferin substrates in the second section. Next, we will highlight several examples that are distinct from conventional analyte sensing designs. Finally, we will provide our outlook and perspective on this exciting field.

2. ABS Probes Based on the Caging of Luciferin

Given that luciferin is the natural substrate of the firefly luciferase (Fluc), it is not surprising that it remains the most common scaffold used in the field for the development of activity-based bioluminescence probes. The recent examples we highlight below can be divided into enzyme- or non-enzyme-activated probes.

2.1. Bioluminescence Probes for Enzymatic Systems

Tumors possess the remarkable ability to evade the immune system. Cancer immunotherapy is an emerging treatment option that utilizes immune checkpoint inhibitors (ICIs) to inhibit programmed cell death ligand 1 (PD-1) [19–21]. This in turn results in the activation of T lymphocytes which releases granzyme B, a serine protease, to induce the apoptosis of cancer cells [22,23]. The ability to visualize granzyme B activity can therefore be used as a proxy to monitor the effectiveness of immunotherapy [24]. To this end, Rao and coworkers [25] developed two activity-based BL probes to report on the activity of granzyme B (Figure 1a). The first, GBLI1, features a tetrapeptide substrate (Ile-Glu-Phe-Asp) appended to aminoluciferin through an amide bond. The second, GBLI2, utilizes a self-immolative linker to enable attachment of the peptide substrate to D-luciferin. Owing to higher overall sensitivity of GBLI2 it was employed in vivo to evaluate the response of CT-26 murine colorectal carcinoma tumors toward ICI. Interestingly, after the animals were ‘cured’, further implantation of CT26-luc colorectal cells did not result in the growth of new tumors. Moreover, the granzyme B to Dluc bioluminescence ratio increased as a function of time (Figure 1b). This result indicates the T cell population was primed to clear the newly implanted cells and that they retain memory of past immune system activation. On the other hand, when a second group of ‘cured’ mice were implanted with 4T1-luc breast cancer cells, the granzyme B to Dluc bioluminescence ratio decreased and new tumors grew (Figure 1b).

Figure 1.

Figure 1.

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a) Schematic representing the sequential reaction between GBLI-1 or GBLI-2 with granzyme-B and luciferase to produce bioluminescence. b) The ratios of GzmB to Dluc signals upon rechallenging cured mice with CT26-luc cells (top) or 4T1-luc cells (bottom). c) Schematic showing the release of aminoluciferin upon sequential activation of BL-FAP by FAP and luciferase. d) Representative images of mice treated with a vehicle control, BL-FAP, or BL-FAP + inhibitor. Figure 1b was reprinted (adapted) with permission from Cell Chemical Biology 2022, 29, 1556–1567. Copyright (2022) Elsevier. Figure 1d was reprinted (adapted) with permission from Analytical Biochemistry 2022, 655, 114859. Copyright (2022) Elsevier.

Timely and non-invasive detection of cancer is crucial in alleviating the disease at the early stage. In this regard, fibroblast activation protein a (FAPa) has emerged as a tumor biomarker as it has been observed in more than 90% of all human cancers [26]. FAPa is a serine protease that plays an essential role in the development and metastasis of cancer cells by degrading extracellular matrix components [27,28]. To detect FAPa activity, Zhang and coworkers [29*] have developed BL-FAP, an activity-based bioluminescence probe featuring N-acetyl-Gly-Pro-OH (Ac-Gly-Pro-OH) appended to aminoluciferin (Figure 1c). In vitro characterization demonstrated that BL-FAP is highly sensitive boasting a LOD of 18.1 pg/mL. Furthermore, it was found to be selective over close analogs such as dipeptide peptidase IV and prolyl oligopeptidase. Finally, studies in mice bearing MGC-803-luc tumors demonstrated robust probe activation, which could be attenuated when the animals were pretreated with SP-13786, a FAP inhibitor (Figure 1d). Beyond this in vivo experiment, the authors also utilized BL-FAP to quantitatively compare the bioluminescence signals from plasma of human patients with gastric cancer, a control healthy subject and a subject with benign gastric lesions to observe that the gastric cancer patients had significantly higher levels of FAPa activity.

2.2. Bioluminescence Probes for Small Molecule Analytes

Norepinephrine (NE) is an important neurotransmitter and hormone utilized by the body to mediate a variety of processes including the ‘flight or fight’ response when it encounters danger [30,31]. Once activated in this context, the body is primed to respond to the perceived stressful situation. However, if the levels of NE do not return to basal levels and remain chronically elevated, it can result in pathological consequences such as high blood pressure and in some instances, the development of cancer [31]. Thus, non-invasive detection of NE levels via in vivo imaging can help to monitor changes in response to various stimulants. To achieve this goal, Feng, Ke, Li and coworkers [32] developed NBP, an activity-based bioluminescence probe for NE detection which contains a responsive carbonothioate trigger [33,34]. Sensing of NE involves initial nucleophilic attack of the NE amino group onto the trigger which displaces the thiophenol to form a carbamate moiety. Subsequently, the NE benzyl alcohol undergoes an intramolecular cyclization event to liberate luciferin (Figure 2a). Of note, the authors did not observe off-target response with other neurotransmitters including epinephrine or dopamine. To test the in vivo performance and NE sensing capabilities of NBP in vivo, the authors utilized FVB-luc+ mice, which were either pretreated with NE or fluoxetine, followed by intravenous injection of NBP (Figure 2b and 2c). The latter compound is a selective serotonin uptake inhibitor, which has been shown to acutely elevate the extracellular concentration of norepinephrine in the brain. As anticipated, the authors noted a marked increase in the bioluminescence signal for both treatment conditions.

Figure 2.

Figure 2.

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a) Schematic illustrating the reaction between NBP with NE to afford luciferin, which further reacts with luciferase to produce bioluminescence. b) Representative bioluminescence images of transgenic mice pre-treated with a vehicle control or fluoxetine (100 mg/mL). ROI represents brain region. c) Quantification of bioluminescence from transgenic mice treated with a vehicle control or NE. d) Schematic illustrating the reaction between P Probe and superoxide anion. e) In vitro assay demonstrating 18.5-fold signal enhancement upon treatment of P Probe with O2.−. f) Representative images of mice treated with PBS and cisplatin at 2 or 4 mg/mL, prior to P Probe administration. Figure 2b and 2c were reprinted (adapted) with permission from Anal. Chem. 2022, 94, 6441−6445. Copyright (2022) American Chemical Society. Figure 2e and 2f were reprinted (adapted) with permission from Biosensors and Bioelectronics 2022, 216, 114632. Copyright (2022) Elsevier.

Cisplatin is an FDA approved drug and is generally used as a first line chemotherapeutic agent [35]. Upon binding to DNA, the ensuing damage results in mitochondria-assisted cell apoptosis. Additionally, it stimulates dinucleotide phosphate (NADPH) oxidases (NOXs) to produce reactive oxygen species (ROSs), such as the superoxide anion (O2.−) which contributes to cancer cell killing [36–39]. Unfortunately, off-target production of O2.− due to cisplatin treatment can result in severe oxidative stress and damage to healthy tissue [40]. As such, selective monitoring of this ROS in vivo is critical. To address this, Du, Song and coworkers [41] have developed an activatable and selective probe for O2.−detection by caging the hydroxyl group of luciferin with a diphenylphosphinyl-based trigger [42]. The resulting probe, known as ‘P probe’, was shown to respond to cisplatin-induced generation of O2.− and further, that minimal cross-reactivity was observed with OCl−, ONOO−, or 1O2 (Figure 2d and 2e). Finally, the authors demonstrated the P probe was efficacious in vivo. When C-38-fLuc tumor-bearing BALB/c mice were treated with a therapeutic dosage level of cisplatin, administration of the P probe resulted in a robust bioluminescence signal enhancement (Figure 2f).

3. Near-infrared Emitting ABS Bioluminescence Probes

Native firefly luciferin-based systems emit green light (λem = 530 nm), which limits the depth penetration owing to the scattering and absorption of photons. Efforts made towards the development of NIR luciferin analogs have resulted in substrates such as AkaLumine, BL660, infraluciferin, OH-pLH2 and CouLuc-3 [43**]. Because there is less interference from endogenous pigments in this imaging window, the use of these substrates can facilitate deep tissue imaging with enhanced sensitivity and signal-to-noise ratios [4,6]. Several ABS probes for NIR bioluminescence imaging have recently been reported and will be discussed below.

Certain diets such as those that are highly processed or contain large quantities of saturated fats have been linked to chronic inflammatory states which in turn may exacerbate tumor progression in humans [44,45]. Nitric oxide (NO) is a key biomarker of inflammation; however, it is challenging to detect in the context of cancer owing to its low steady state concentration (<200 nM), high reactivity, and fleeting nature [46,47]. To address this, Chan and coworkers [48**] turned to bioluminescence imaging by developing the first ABS probe (BL660-NO) for NO sensing in the NIR window. BL660-NO features an NO-responsive o-phenylenediamine trigger installed onto the carboxylate group of BL660 [49]. The ensuing reaction with NO (via N2O3) was designed to be rapid to ensure high sensitivity. Subsequently, rate-limiting hydrolysis of the acyl triazole intermediate liberates the substrate which can then react with luciferase to produce bioluminescence (λem = 660 nm) (Figure 3a). To elucidate the connection between diet, inflammation, and cancer, the authors designed two longitudinal studies where animals were fed either low-fat or high-fat diets for 12 or 24 weeks before 4T1-Luc murine breast cancer cells were implanted into the mammary fat pads (Figure 3b). Interestingly, in both sets of experiments the animals that were fed a high-fat diet grew notably larger tumors. Additionally, bioluminescence imaging with BL660-NO revealed: 1) all tumors from both high-fat groups were more inflamed as indicated by a stronger bioluminescence signal and 2) subjecting animals to high fat for a longer duration resulted in a more pronounced inflammatory state (turn on response of 2.6-fold vs. 14.3-fold) (Figure 3c).

Figure 3.

Figure 3.

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a) Schematic illustrating NO-mediated activation of BL660-NO. b) Experimental design showing mice on high-fat or low-fat diets being implanted with 4T1-luc followed by administration of BL660-NO. c) Quantified data of bioluminescence signals from the 24-week diet study. d) Schematic illustrating activation of BL660-NTR via nitroreductase activity. e) Representative images of mice showing activation of the trigger compared to signal due to background hydrolysis. Figures 3b and 3c were reprinted (adapted) with permission from ACS Cent. Sci. 2022, 8, 461−472. Copyright (2022) American Chemical Society. Figures 3d and 3e were reprinted (adapted) with permission from J. Am. Chem. Soc. 2023, 145, 1460−1469. Copyright (2023) American Chemical Society.

Tumor hypoxia represents another important biomarker that is often associated with poor clinical prognosis [50]. This condition is defined as a state where the supply of oxygen is insufficient to meet the requirements of the tissue. The ability to reliably detect hypoxia in the preclinical setting is desirable because this condition can lead to drug and radiotherapy resistance. A common approach to visualize tumor hypoxia is to exploit the enzymatic activation of aryl nitro triggers by nitroreductase (NTR) activity under oxygen deficient conditions [50]. Initial attempts by Chan and coworkers [51*] to install a trigger for NTR onto BL660 via a standard self-immolative linker was not successful owing to spontaneous hydrolysis and off-target esterase cleavage. This prompted the authors to design a new hydrolysis-resistant linker featuring an isopropyl shielding arm to stabilize the adjacent ester. This new technology ultimately enabled the development of BL660-NTR, an ABS probe for detection of NTR activity (Figure 3d). In vitro evaluation revealed that only ~1% of BL660-NTR hydrolyzed after incubation in PBS for 24 hour at 37 °C, whereas over 28% of the congener equipped with the standard linker was cleaved. The authors observed an even more striking difference when the two probes were treated with esterase. After one hour, 1.9% and 78.7% of BL660-NTR and the primary alcohol ester analog were consumed, respectively. To evaluate BL660-NTR in vivo, the authors established a murine model of lung cancer using human A549 cells. Within minutes after systemic administration of the probe, the authors observed a significant signal enhancement in the tumor sites, indicating the presence of NTR activity (Figure 3e).

4. Beyond Conventional ABS Bioluminescence Probes

Beyond the detection of the various enzyme and small molecule-based biomarkers, we highlight several probe systems for nonconventional sensing applications.

For instance, with impressive advances in the field of synthetic biology, it is possible to engineer bacteria that can produce various therapeutics in vivo, including those for cancer treatment [52,53]. However, it is not trivial to monitor where these bacteria are localized to within the body or whether they have successfully targeted the tumor site. To address this challenge, Jiang et al. [54] developed a bioluminescent strategy to image bacteria in vivo over multiple days. Specifically, the authors employed chromosome integration to introduce Fluc and luciferin regenerating enzyme (LER) into a probiotic E. coli strain known as Nissle 1917 (EcN) (Figure 4a). After luciferin is converted to oxyluciferin by Fluc, LER is able to transform it into 2-cyano-6-hydroxybenzothiazole which in the presence of D-cysteine will regenerate an equivalent of luciferin (Figure 4b). The authors proposed that luciferin recycling would be critical to yield a persisting bioluminescence signal in vivo. To evaluate their approach, the authors generated a 4T1 breast tumor model. One group of mice were injected with PBS (control) and the other was treated with the engineered bacteria (EcN-Fluc-LRE) via intravenous administration. Subsequently, D-luciferin and D-cysteine were injected intratumorally and imaged. A strong and persistent bioluminescence signal was only observed in the bacteria-treated group, indicating successful targeted the tumor site (Figure 4c).

Figure 4.

Figure 4.

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a) Cartoon representation of mice harboring mutant bacteria containing Fluc-LRE genes. b) Schematic representing reactions of luciferin with Fluc and regeneration of the substrate in presence of LRE and D-cysteine. c) Representative bioluminescence images of mice harboring the mutant bacteria. d) Structure of masked bioluminescence substrate ETZ. e) Structural representation of mutant enzyme BRIC. f) Representative images of live mice with the hippocampus transduced with BRIC or OCaMBI110 AAVs. Figures 4a, 4b and 4c were reprinted (adapted) with permission from Anal. Chem. 2021, 93, 15687−15695. Copyright (2021) American Chemical Society. Figures 4e and 4f were reprinted (adapted) with permission from Nat. Commun. 2022, 13, 3967. Copyright (2022) Springer Nature.

Of note, the authors were also successful in utilizing this technology to study the digestive track of mice by feeding recombinant bacteria via oral gavage and subsequently dosing with D-luciferin and D-cysteine.

In another example, non-invasive imaging of calcium (Ca2+) transients in the brain is a long-standing challenge. Despite the emergence of probes designed for various modalities, bioluminescence imaging of the brain is a powerful alternative owing to high sensitivity of this approach and absence of background interference. Beyond the substrate and luciferase pairs highlighted above, the use of bioluminescent systems based on the coelenterazine scaffold (Figure 4d) has recently gained momentum due to the fact that light generation is ATP-independent and further, the rate of reaction is faster which results in a higher volume of light production relative to insect luciferin-luciferase pairs. However, most coelenterazine systems emits blue light which inevitably limits tissue penetration. In this regard, Ai and coworkers developed a mutant luciferase teLuc and coupled it to a red fluorescent protein (mScarlet-I) to obtain Bioluminescent Red Protein (BREP) which emits above 600 nm [55**]. Moreover, the authors inserted CaM and M13 in teLuc between residues 133 and 134 to obtain Bioluminescent Red Indicator for Ca2+ (BRIC) (Figure 4e). This system was characterized in vitro using a new prosubstrate coined ETZ (Figure 4d) by caging DTZ at the C3 position with a succinate group. The incorporation of succinate enhanced solubility, uptake through blood-brain barrier (BBB) and prevented the P-glycoproteins (which acts as a BBB efflux pump) from pumping the molecule out. Upon gaining cell entry, ETZ can be converted to DTZ nonspecifically by intracellular esterases. To test their system in vivo, the authors systemically injected ETZ to detect the neuronal activity in mice brain that expressed BRIC via Ca2+ imaging. Additionally, the authors demonstrated that upon comparing BRIC with OCaMBI110 [56] with their respective substrates, the bioluminescence signal from the BRIC-ETZ pair was significantly higher (Figure 4f).

4. Conclusion and Future Outlook

Because external excitation is not required to produce light output, bioluminescence imaging is one of the preferred modalities to achieve high sensitivity in vivo. By leveraging this notable advantage, probe developers have utilized various caging strategies to develop activity-based sensing probes and have applied these specialized molecules to study a diverse palette of biomarkers in the context of both health and disease states. As discussed above, such probes have traditionally been based on the luciferin scaffold which remains the most widely used system in the field (Section 2). However, it is notable that new examples have recently emerged that have taken advantage of red-shifted substrate and enzyme pairs to achieve greater tissue penetration (Section 3). An exciting application will be to simultaneously employ probes belonging to each of these classes to examine potential crosstalk between related systems via multiplex bioluminescence imaging. Finally, in Section 4 we have discussed several examples that extend beyond conventional probe designs such as those that entail substrate caging. With the diversity of targets and the creative use of bioluminescence, it will be exciting to see what major breakthroughs will be achieved in the near future.

Acknowledgements

The authors acknowledge the National Institutes of Health for funding (R35GM133581).

Footnotes

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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