Critical angle reflection imaging for quantification of molecular interactions on glass surface
Authors: Guangzhong Ma
Shaopeng Wang
Runli Liang
Zijian Wan
Journal: Nature Communications (2021)
Discipline: Multidisciplinary
Keywords: Surface plasmon resonance; molecular interactions; label-free techniques; sensitivity; refractive index change
Affiliations: Biodesign Center for Biosensors and Bioelectronics, Arizona State University, Tempe, AZ, USA., Biodesign Center for Biosensors and Bioelectronics, Arizona State University, Tempe, AZ, USA. .

Abstract:  Quantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR). The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near the critical angle. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. CAR imaging can be performed on SPR imaging setups. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR performs, including proteins, nucleic acids and cell-based detections. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. CAR exhibits several distinct characteristics, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. We anticipate CAR can expand SPR's capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry. 

Retracted: No

PubPeer comments:
 To the authors or anyone else interested This article got my attention as it seems to claim that the critical angle can be used for surface-based sensing. Overall, this is a bit confusing to me since it is very well established that the TIR angle depends on bulk properties, not surface binding. (This is why it the TIR angle is such a useful complementary parameter in conventional SPR.) I would like to make some comments. As always, my intention is simply to have an open discussion on some points that I fail to understand and I hope the authors do not see it as something negative that their work is discussed. I believe surface binding to glass will cause minor reflectivity changes and they will of course appear also in the TIR angle region of the angular spectrum. This would be similar to the iSCAT method introduced by Piliarik and Sandoghdar 2014 (cited). Do I understand it right that this kind of interferometry is essentially what the authors are measuring in the surface binding experiements? In other words, a reflectivity change is detected rather than a *shift* in the TIR angle? (Although a shift is of course expected and observed when changing the bulk refractive index.) I would also like to know if any control experiments were performed when it comes to the detection of antibodies and small molecules? The most straghtforward would be to introduce the same analytes but without any receptors on the surface. Also, one could introduce another antibody or small molecule that is not expected to bind to the receptors on the surface. I would be curious to know what the sensorgrams look like then? Thanks for your attention! Andreas ,