The primary application of 1-Pyrenebutyric acid NHS ester is as a labeling reagent for biomolecules containing primary amines (-NH₂). Its mechanism of action is based on the reactivity of the N-hydroxysuccinimide (NHS) ester group with primary amines to form stable amide bonds. The NHS ester reacts rapidly and selectively with amines under mild conditions (pH 7-9), resulting in the covalent attachment of the pyrene fluorophore to the target molecule. This allows for the sensitive detection and tracking of the labeled biomolecule using the strong fluorescence of the pyrene moiety.

2,5-Dioxopyrrolidin-1-yl 4-(pyren-1-yl)butanoate is an amine-reactive ester that has been widely used for coating carbon nanotube-based biosensors for the capture of antibodies and proteins.
In vitro, 1-Pyrenebutyric acid NHS ester is used as an aromatic reagent for biomolecule labeling. It is a linker that may be utilized to prepare electrochemical biosensors. The compound is often used as an activating reagent for carboxylic acids in organic chemistry or biochemistry. Its NHS ester functionality enables covalent conjugation to amine-containing substrates, supporting applications in nucleic acid detection, protein immobilization, and characterization of cyclodextrin-based polyrotaxanes.

In vivo activity of 1-Pyrenebutyric acid NHS ester is not typically studied, as it is a labeling reagent used to modify biomolecules in vitro. However, the labeled biomolecules (e.g., labeled antibodies or proteins) could be used in vivo for imaging or tracking purposes. The pyrene fluorophore provides bright blue emission that can be detected in tissues, allowing researchers to study the distribution and dynamics of the labeled molecules.

In vitro enzyme/receptor binding (non-cell) assays using 1-Pyrenebutyric acid NHS ester typically involve the compound as a label for biomolecules. A standard protocol for labeling a protein: the protein is dissolved in a buffer (e.g., 0.1 M sodium bicarbonate, pH 8.5). A solution of 1-Pyrenebutyric acid NHS ester in DMSO is added to the protein solution. The reaction mixture is incubated at room temperature for 1-2 hours in the dark. The labeled protein is purified by dialysis or size exclusion chromatography. The degree of labeling is determined by measuring the absorbance at 342 nm (pyrene) and 280 nm (protein).

In vitro cell-based assays using 1-Pyrenebutyric acid NHS ester typically involve using the compound to label cell surface proteins or to detect specific biomolecules. A standard protocol for labeling cell surface proteins: cells are harvested and incubated with a 1-Pyrenebutyric acid NHS ester-labeled antibody in PBS for 30-60 minutes at 4°C. Cells are washed to remove unbound antibody and analyzed by flow cytometry. The fluorescence intensity measured corresponds to the expression level of the target protein on the cell surface. Alternatively, the labeled antibody can be used in immunofluorescence microscopy to visualize the target protein.

In vivo animal experiments for 1-Pyrenebutyric acid NHS ester are typically not performed for the compound itself, but for 1-pyrenebutyric acid-labeled biomolecules. A standard protocol for biodistribution of a labeled antibody: a 1-pyrenebutyric acid-labeled antibody is administered intravenously to mice. At various time points, animals are euthanized, and organs are collected. The tissues are homogenized, and the fluorescence intensity is measured using a fluorometer to determine the distribution of the antibody. Alternatively, whole-body imaging can be performed to visualize the localization of the labeled antibody in real-time.

Pharmacokinetic properties of 1-Pyrenebutyric acid NHS ester are not a primary focus of study. The compound is a reactive labeling reagent that is typically used in vitro. Once conjugated to a biomolecule, the pharmacokinetics of the conjugate are determined by the biomolecule rather than the dye. The compound is typically stored at -20°C. Its reactivity with water (hydrolysis) is a consideration for its handling and use.

Toxicity data for 1-Pyrenebutyric acid NHS ester is limited. As a chemical reagent, it should be handled with standard laboratory precautions. The NHS ester group is reactive and can cause skin and eye irritation. Inhalation, ingestion, and skin contact should be avoided. Appropriate personal protective equipment should be worn. The compound is not intended for human use.

[1]. Benvidi A, et al. Comparison of impedimetric detection of DNA hybridization on the various biosensors based on modified glassy carbon electrodes with PANHS and nanomaterials of RGO and MWCNTs. Talanta. 2016 Jan 15;147:621-7.
[2]. Tian J, et al. Biosensing platform based on graphene oxide via self-assembly induced by synergic interactions. Anal Biochem. 2014 Sep 1;460:16-21.
[3]. Kim, J.P., Lee, B.Y., Hong, S., et al. Ultrasensitive carbon nanotube-based biosensors using antibody-binding fragments. Anal. Biochem. 381(2), 193-198 (2008).
[4]. Karachevtsev, V.A., Stepanian, S.G., Glamazda, A.Y., et al. Noncovalent interaction of single-walled carbon nanotubes with 1-pyrenebutanoic acid succinimide ester and glucoseoxidase. J. Phys. Chem. 115(43), 21072-21082 (2011).

λmax: 234, 243, 265, 276, 326, 342 nm
Emission: 377; 397

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1. Chemically Functionalized Graphene Field-Effect Transistor Biosensor for Label-Free Exosome Detection
Graphene field-effect transistors (gFETs) were non-covalently functionalized with 1-pyrene butyrate N-hydroxysuccinimide and conjugated with an anti-CD63 antibody for label-free exosome detection. A portion of the graphene film was exposed to solution using a microfluidic channel. The changes in the electrical properties of the exposed graphene were visualized in the drain-source current (Ids) vs. back-gate voltage (Vg) curves, showing a new minimum value next to the original Dirac point. An additional minimum value appeared at Vglower in the presence of phosphate-buffered saline (PBS), located outside the original Dirac point, and this minimum value shifted over time as exosomes were introduced into the channel. This minimum deviation relative to the PBS reference point saturated after 30 minutes and was observed at various exosome concentrations. When conjugated with an isotype control antibody, the sensor's response to the highest concentration of exosomes was negligible compared to that of the anti-CD63 antibody, indicating that the functionalized gFET can specifically detect exosomes at concentrations as low as 0.1 μg/mL and is concentration-sensitive. This gFET biosensor, previously unused for exosome detection, could be an effective liquid biopsy tool for detecting exosomes as biomarkers for early disease identification, such as cancer.
Reference: Sci Rep. 26 Sep 2019; 9(1):13946. doi: 10.1038/s41598-019-50412-9.

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2. A Novel Electrochemical DNA Biosensor Based on Fe3O4NPs-Reduced Graphene Oxide/PANHS Nanocomposite Modified Magnetic Rod-Shaped Carbon Paste Electrode
This study designed a label-free DNA biosensor based on a magnetic rod-shaped carbon paste electrode (MBCPE). This electrode was modified with Fe3O4/reduced graphene oxide (Fe3O4NP-RGO) nanocomposite and 1-pyrenebutyrate-N-hydroxysuccinimide ester (PANHS) binder for DNA sequence detection. The probe (BRCA1 5382 insC mutation detection) strand was immobilized on the MBCPE/Fe3O4-RGO/PANHS electrode under precise incubation time. The modified electrode was characterized using scanning electron microscopy (SEM), infrared spectroscopy (IR), vibrating sample magnetometer (VSM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry. Experimental parameters such as probe DNA immobilization time, hybridization time, and temperature were investigated. Under optimal conditions, probe immobilization and hybridization with target DNA (complementary DNA) were tested. This DNA biosensor exhibited a good linear relationship between ΔRct and logarithm within a complementary target DNA concentration range of 1.0 × 10⁻¹⁸ mol L⁻¹ to 1.0 × 10⁻⁸ mol L⁻¹, with a correlation coefficient of 0.9935 and a detection limit of 2.8 × 10⁻¹⁹ mol L⁻¹. Furthermore, this biosensor was successfully applied to distinguish between complementary and non-complementary sequences. The constructed biosensor (MBCPE/Fe3O4-RGO/PANHS/ssDNA) possesses high sensitivity, high selectivity, high stability, high reproducibility, and low cost, and can be used to detect BRCA1 5382 insC mutations.
Reference: Mater Biol Appl. 2016 Nov 1;68:1-8. doi: 10.1016/j.msec.2016.05.056.

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3. Detection of Alpha-Fetoprotein in Plasma from Hepatocellular Carcinoma Patients Using Graphene Field-Effect Transistors
Detection of alpha-fetoprotein (AFP) in plasma is crucial for the diagnosis of human hepatocellular carcinoma (HCC). We developed a biosensor to detect AFP in plasma and phosphate-buffered saline (PBS) from HCC patients using graphene field-effect transistors (G-FETs). G-FETs were functionalized with 1-pyrenebutyrate N-hydroxysuccinimide ester (PBASE) to immobilize anti-AFP antibodies. AFP was detected by evaluating the shift in Dirac point voltage (ΔVDirac) after AFP bound to the surface of the G-FET channel immobilized with anti-AFP antibodies. The G-FET biosensor immobilized with anti-AFP antibody was able to detect AFP at a concentration of 0.1 ng mL⁻¹ in PBS buffer with a detection sensitivity of 16.91 mV. In HCC patient plasma, the biosensor was able to detect AFP at a concentration of 12.9 ng mL⁻¹ with a detection sensitivity of 5.68 mV. The sensitivity (ΔVDirac) depended on the concentration of AFP in the PBS buffer or HCC patient plasma. These data indicate that the G-FET biosensor has practical application value in the diagnostic field. November 19, 2018; 18(11): 4032. doi: 10.3390/s18114032.
1-Pyrenebutyric acid NHS ester is a linker and activating reagent for carboxylic acids in organic chemistry or biochemistry. It is used for biomolecule labeling. The NHS ester enables covalent conjugation to amine-containing substrates. It is also used to prepare electrochemical biosensors. It is not a drug and has no clinical use or approval status. It is commercially available from chemical suppliers for research purposes only.

C24H19NO4

385.4120

385.131

C, 74.79; H, 4.97; N, 3.63; O, 16.60

114932-60-4

130767

Typically exists as light green to green solids at room temperature

1.4±0.1 g/cm3

590.9±43.0 °C at 760 mmHg

132-136ºC(lit.)

311.2±28.2 °C

0.0±1.7 mmHg at 25°C

1.739

3.77

0

4

6

29

660

0

O(C(C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C2C([H])=C([H])C3C([H])=C([H])C([H])=C4C([H])=C([H])C=1C=2C4=3)=O)N1C(C([H])([H])C([H])([H])C1=O)=O

YBNMDCCMCLUHBL-UHFFFAOYSA-N

InChI=1S/C24H19NO4/c26-20-13-14-21(27)25(20)29-22(28)6-2-3-15-7-8-18-10-9-16-4-1-5-17-11-12-19(15)24(18)23(16)17/h1,4-5,7-12H,2-3,6,13-14H2

(2,5-dioxopyrrolidin-1-yl) 4-pyren-1-ylbutanoate

114932-60-4; 1-Pyrenebutyric acid N-hydroxysuccinimide ester; 2,5-DIOXOPYRROLIDIN-1-YL 4-(PYREN-1-YL)BUTANOATE; N-Hydroxysuccinimidyl Pyrenebutanoate; 97427-71-9; Pyrenebutyric acid NHS ester; 1-Succinimidyl-3'-pyrenebutyrate; 1-Pyrenebutanoic acid, 2,5-dioxo-1-pyrrolidinyl ester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~259.46 mM)
DMF: ~15 mg/ml

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)