Nanomedicine. 2017 Feb;13(2):383-390. doi: 10.1016/j.nano.2016.08.014.

A nanobiosensor for the detection of arginase activity.

Malalasekera AP1, Wang H1, Samarakoon TN1, Udukala DN1, Yapa AS1, Ortega R1, Shrestha TB2, Alshetaiwi H2, McLaurin EJ1, Troyer DL2, Bossmann SH3.

1Department of Chemistry, Kansas State University, Manhattan, KS, USA.
2Department of Anatomy & Physiology, Kansas State University, Manhattan, KS, USA.
3Department of Chemistry, Kansas State University, Manhattan, KS, USA. Electronic address: sbossman@ksu.edu.

 

Abstract

A nanobiosensor for arginase detection was designed and synthesized. It features a central dopamine-coated iron/iron oxide nanoparticle to which sulfonated cyanine 7.0 is tethered via a stable amide bond. Cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. Based on calibration with commercially obtained arginase II, the limit of detection (LOD) is picomolar. It is noteworthy that the nanobiosensor for arginase detection does not show a fluorescence increase when incubated with the enzyme NO-reductase, which also uses arginase as substrate, but is indicative of an inflammatory response by the host to cancer and infections. Arginase activity was determined in a syngeneic mouse model for aggressive breast cancer (4T1 tumors in BALB/c mice). It was found that the arginase activity is systemically enhanced, but especially pronounced in the active tumor regions. Copyright © 2016 Elsevier Inc.

KEYWORDS:

Arginase detection; Iron/iron oxide nanoparticle-based nanoplatform; Posttranslational sensor; Quantitative fluorescence detection

PMID: 27558349

 

Supplement:

Arginase metabolizes L-arginine to L-ornithine and urea.  Besides its fundamental role in the hepatic urea cycle, arginase is a key player in the immune system. In humans, arginase I is constitutively expressed in polymorphonuclear neutrophils, released during inflammation and responsible for the down-regulation of nitric oxide synthesis by converting arginine to ornithine. Arginase-mediated L-arginine depletion is capable of suppressing T cell immune responses, leading to inflammation-associated immune-suppression, which is a hallmark of aggressive solid tumors.[2] Furthermore, L-arginine insufficiency is also responsible for dysfunction of natural killer (NK) cells, which are vital for early host defense against infections and tumors.[3] Arginase can be found in mammalian bodies in two isoforms: arginase I and arginase II. Arginase I has been recognized as a biomarker for pancreatic adenocarcinoma.[4] Arginase II is expressed in cancer-associated fibroblasts and indicates tissue hypoxia and predicts poor outcomes in patients with pancreatic cancer.[5] However, since fast, reliable and sensitive plate reader tests for arginase detection are not available to date, numerous potential other applications for this technology can be envisioned, such as the detection of immune responses after trauma or surgery, as well as autoimmune disorders.[6] Colorimetric arginase tests kits are currently commercially available. The most sensitive test to data has a limit of detection (LOD) of 2 x 10-7 moles L-1. [7] In this UV/Vis-absorption-based assay, arginase reacts with arginine and undergoes a series of reactions to form a colored product (lmax= 570 nm). The aim of this endeavor is to design a fluorescence-based arginase sensor that is equally sensitive as the technically more complex immunoassays (target LOD: sub-picomolar).[8]

 

It is noteworthy that the transformation of arginine into ornithine proceeds stepwise and most likely in a random pattern. It ends when all seven arginine units in GRRRRRRRG (pI: 12.78)[9] have been transformed into GOOOOOOOG (pI: 5.52). Assuming a random reaction pattern also means that 176 different reaction intermediates can be formed, before the reaction stops at GO7G.

 

The posttranslational arginase sensors utilize cyanine dyes as FRET (Förster Resonance Energy Transfer) probes.[10] Cyanine 5.5 (donor) and cyanines 7.0/7.5 (acceptors) form attractive FRET-pairs, as indicated in Scheme 1, because of the significant spectra overlap between the fluorescence spectrum of cyanine 5.5 and the absorption spectra of both, cyanine 7.0 and 7.5. Furthermore, all cyanine dyes are characterized by their large molecular extinction coefficients and their narrow absorption and emission bands.[11] It should be noted that cyanine dyes have very short fluorescence lifetimes in the range of 200 to 350 ps in water.[12] Therefore, they are essentially not quenched by oxygen. Cyanine dyes have reasonable fluorescence quantum yields ranging between 5 and 10 percent in aqueous buffers and in-vivo.[13]

 

 

Scheme 1: Spectral overlap between the potential Förster Resonance Transfer pairs: Cyanine 5.5/Cyanine 7.0 and Cyanine 5.5/Cyanine 7.5. The absorption and emission spectra of the cyanines are normalized. From this estimation, the spectral overlap in both FRET pairs appears to be of the same order of magnitude. Information downloaded from www.lumiprobe.com was utilized in preparing this scheme.

 

Figure 2: The authors with other lab members: front row from left to right: Dr. Aruni P. Malalasekera, Raquel Ortega, Yubisela Toledo, Dr. Asanka S. Yapa. Back row from left to right: Lauren Chlebanowski, Faith Rahman, Dr. Hongwang Wang, Jose Covarrubias, Dr. Stefan H. Bossmann, Jing Yu, Dr. Madumali Kalubowilage, Obdulia Covarrubias-Zambrano.

 

References

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