Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

Abstract

Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans.

Surface-Enhanced Raman Spectroscopy-Based Label-Free Insulin Detection at Physiological Concentrations for Analysis of Islet Performance

“…[51][52] And, pancreatic polypeptide does not show the Raman peak at 1002 cm -1 from phenylalanine. 53 Hence, when detecting insulin in islet secretion using the peak at 1002 cm -1 , the influence of other hormones on our measurements can be ignored. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 11…”

A Pharmacokinetic Model of a Tissue Implantable Insulin Sensor

“…Additional approaches utilize QCM transducers and molecularly imprinted polymers either by insulin or an insulin antibody; however the detection limit is in the mM range [17] . Alternative methods include imaging ellipsometry of an insulin antibody modified gold surface [18] , fluorescent imaging of fluorescently labeled insulin binding aptamer immobilized on graphene oxide [19] , and hyperspectral Raman spectroscopy of human islets of Langerhans [20] .…”

Glucagon – the new ‘insulin’ in the pathophysiology of diabetes

“…Given the growing interest in understanding the role of glucagon in the pathophysiology of diabetes, it is not surprising that effort was devoted to developing tools to study glucagon and glucagon regulation including the justification and validation of new sensitive and specific assays for glucagon detection [110][111][112][113]. Another area of interest was development of system biology tools incorporating glucagon action and regulation.…”