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D-Luciferin (sodium salt)

Catalog No.GC43497

D-Luciferin (sodium salt) Chemical Structure

D-Luciferin sodium salt is the substrate of luciferases that catalyze the production of light in bioluminescent insects.

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Cell experiment [1]:

Cell lines

B16-F10 and MCF-7 clonal cells

Preparation Method

Cells that stably express luciferase were seeded in 12-well plates (2 × 103/well). Cells were treated with D-luciferin substrate diluted in media or media alone.

Reaction Conditions

30 min incubation at 37°C in the dark, washed with PBS once and replaced the normal growth medium.


D-luciferin could be used to investigate whether biophotonic activity or the luciferase gene itself have negative influence on cell growth in vitro. Luciferase bioluminescence was not sufficient to generate photodynamic toxicity in vitro. It has little difference of cell viability between clones expressing different levels of luciferase or between cells periodically treated with D-luciferin.

Animal experiment [2]:

Animal models

BALB/C nude (females), aged 6–8 weeks

Preparation Method

HEK 293T cells were prepared at a concentration of 2 × 106 cells/ml in PBS; Injecting 1 × 105 HEK 293T cells into both flanks of mice immediately, images were acquired after injection of D-luciferin.

Dosage form

150 mg/kg


The BLI measurements were performed when the peak of emission occurred, after injection of the luciferins into animals. Luc2/ D-luciferin, CBG99/ D-luciferin, CBR2/ D-luciferin, and Akaluc/ D-luciferin produced peaks at 610 nm, 540 nm, 620 nm, and 640 nm, respectively. When CycLuc1 was used as a substrate, the emission peak of Luc2 and Akaluc were green shifted towards 600 nm. Analysed the total emission of each luciferase in vivo with D-Luciferin or the luciferin analogues, Luc2, CBG99, and CBR2 paired with D-luciferin produced the highest signals which were 20-fold higher than that of Akaluc/ D-luciferin (p value < 0.001).


[1]. Tiffen JC,et al. Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo. Mol Cancer. 2010 Nov 22;9:299. doi: 10.1186/1476-4598-9-299.

[2]. Zambito G. Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging. Mol Imaging Biol. 2020 Dec;22(6):1523-1531. doi: 10.1007/s11307-020-01523-7.


D-Luciferin sodium salt is the substrate of luciferases that catalyze the production of light in bioluminescent insects.

D-Luciferin is a common substrate for luciferase and is frequently used in the entire biotechnology field, especially in vivo imaging techniques. The mechanism of action of the imaging is that D-luciferin (substrate) is oxidized to emit light under the action of ATP and luciferase. When D-luciferin is in excess, the number of photons produced is positively correlated with the concentration of luciferase. After transfecting a plasmid carrying a luciferase-encoding gene (Luc) into cells, cells with Luc is engrafted into research animals, and then injected with D-luciferin to detect changes in light intensity through bioluminescence imaging (BLI). The effect of ATP on this reaction system can also be used to indicate energy or vital signs based on changes in bioluminescence intensity. [1][2]

D-luciferin reacts with luciferase, ATP and oxygen with light emission, and the light is detected by a sensitive photographic film.[3] All in vitro D-luciferin measurements were obtained after 1 min at 37 °C using a 30-s acquisition time using a series of filters ranging from 520 to 800 nm. In vitro analysis demonstrated that at a relatively low, but biologically relevant (in vivo) substrate concentration (0.1 mM), three of the four luciferases give maximum signal when combined with D-luciferin. [4]

In vivo analysis showed that the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was D-luciferin. CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2. When paired with CycLuc1 or Akalumine-HCl, Akaluc was brighter. Besides, new combinations of luciferases with distinct colors having potential for multiplexing with a single substrate in superficial tissue were recommended, such as CBG99/ D-luciferin (540 nm) and CBR2/ D-luciferin (620 nm); CBG99/ D-luciferin (540 nm) and Luc2/ D-luciferin (610 nm).[4]

[1] McElroy WD. The Energy Source for Bioluminescence in an Isolated System. Proc Natl Acad Sci U S A. 1947 Nov;33(11):342-5. doi: 10.1073/pnas.33.11.342.
[2] GREEN A, MCELROY WD. Function of adenosine triphosphate in the activation of luciferin. Arch Biochem Biophys. 1956 Oct;64(2):257-71. doi: 10.1016/0003-9861(56)90268-5.
[3] Hauber R. A New, Very Sensitive, Bioluminescence-Enhänced Detection System for Protein Blotting. J. Clin. Chem. Clin. Biochem. 1987,25, pp. 511-514. doi:10.1515/cclm.1987.25.8.511.
[4] Zambito G. Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging. Mol Imaging Biol. 2020 Dec;22(6):1523-1531. doi: 10.1007/s11307-020-01523-7.

Chemical Properties

Cas No. 103404-75-7 SDF
Synonyms N/A
Chemical Name N/A
Canonical SMILES O=C([O-])[C@H]1CSC(C(S2)=NC3=C2C=C(O)C=C3)=N1.[Na+]
Formula C11H7N2O3S2•Na M.Wt 302.3
Solubility DMSO : 100 mg/mL (330.80 mM) H2O : 33.33 mg/mL (110.25 mM) Storage Store at -20°C,protect from light
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Molecular Design of D-Luciferin-Based Bioluminescence and 1,2-Dioxetane-Based Chemiluminescence Substrates for Altered Output Wavelength and Detecting Various Molecules

Molecules2021 Mar 15;26(6):1618.PMID: 33803935DOI: 10.3390/molecules26061618

Optical imaging including fluorescence and luminescence is the most popular method for the in vivo imaging in mice. Luminescence imaging is considered to be superior to fluorescence imaging due to the lack of both autofluorescence and the scattering of excitation light. To date, various luciferin analogs and bioluminescence probes have been developed for deep tissue and molecular imaging. Recently, chemiluminescence probes have been developed based on a 1,2-dioxetane scaffold. In this review, the accumulated findings of numerous studies and the design strategies of bioluminescence and chemiluminescence imaging reagents are summarized.

Luminogenic D-Luciferin Derivatives as OATP1B1 and 1B3 Substrates in No-wash Assays

Photochem Photobiol2021 Nov;97(6):1407-1416.PMID: 33948961DOI: 10.1111/php.13443

The human hepatic organic ion transporting polypeptides OATP1B1 and -1B3 are uptake transporters that influence the disposition of several small molecule drugs and perpetrate certain adverse drug-drug interactions. To predict these in vivo effects, in vitro systems are used to screen new drug entities as potential transporter substrates or inhibitors. To simplify such studies, we synthesized luminogenic derivatives of the OATP1B1 and -1B3 substrate D-Luciferin to test as probe substrates in a rapid, no-wash optical approach for substrate and inhibitor identification and characterization. Each derivative is a pro-luciferin containing a self-immolating trimethyl lock quinone linker that is sensitive to intracellular reducing environments that cause the release of free luciferin in proportion to the amount of probe taken up by the transporter. A subsequent luciferin-limited luciferase reaction produces light in proportion to transporter activity. We tested the derivatives in HEK293 cells that overexpress OATP1B1 or OATP1B3 by transient transfection or viral transduction. Derivatives were identified that showed OATP-dependent uptake that was time and concentration dependent, saturable and sensitive to inhibition by known OATP1B1 and -1B3 substrates and inhibitors. These luminogenic transporter probes enabled an add-only multi-well plate protocol suitable for automation and high throughput screening.

Glucuronidation of D-Luciferin In Vitro: Isoform Selectivity and Kinetics Characterization

Eur J Drug Metab Pharmacokinet2019 Aug;44(4):549-556.PMID: 30820844DOI: 10.1007/s13318-019-00549-9

Background: D-Luciferin is one of the most commonly used substrates in bioluminescence imaging for real-time monitoring of sophisticated biological processes in models of human biology or disease in vitro and in vivo. D-Luciferin is rapidly cleared from blood circulation after being exogenously delivered in vivo and the presence of phenolic groups indicates that glucuronide conjugation is a possible metabolic pathway for the compound.
Objectives: This study aimed to characterize the glucuronidation pathway of D-Luciferin in human liver microsomes (HLM) and human intestine microsomes (HIM).
Methods: HLM and HIM were employed to catalyze the glucuronidation of D-Luciferin in vitro. The activity of recombinant uridine-diphospho-glucuronosyltransferase (UGT) isoforms towards D-Luciferin glucuronidation was screened. Chemical inhibition assay and kinetic analysis was combined to determine the UGT isoforms responsible for the formation of D-Luciferin glucuronide in HLM and HIM.
Results: D-Luciferin could be catalyzed to form one mono-glucuronide which was characterized as 6'-O-glucuronide in HLM and HIM. UGT1A1, 1A3, 1A6, 1A8, 1A9 and 1A10 participated in the formation of D-Luciferin glucuronide, with UGT1A1 exhibiting the highest catalytic activity. Both chemical inhibition assays and kinetic analysis showed that UGT1A1 and UGT1A3 played important roles in D-Luciferin-6'-O-glucuronidation in HLM and HIM, with UGT1A6 also giving a non-negligible contribution to this biotransformation in HLM.
Conclusions: UGT1A1, UGT1A3 and UGT1A6 were responsible for 6'-O-glucuronidation of D-Luciferin in HLM, while UGT1A1 and UGT1A3 were the major contributors to this biotransformation in HIM.

Beyond D-Luciferin: expanding the scope of bioluminescence imaging in vivo

Curr Opin Chem Biol2014 Aug;21:112-20.PMID: 25078002DOI: 10.1016/j.cbpa.2014.07.003

The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive imaging in live mice. However, photon emission from the luciferase is crucially dependent on the chemical properties of its substrate, D-Luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of bioluminescence imaging.

Stability of D-Luciferin for bioluminescence to detect gene expression in freely moving mice for long durations

Luminescence2021 Feb;36(1):94-98.PMID: 32721066DOI: 10.1002/bio.3917

Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate D-Luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of D-Luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l-Luciferin, which is synthesized using racemization of D-Luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that D-Luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.


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