Poster S10 - Elliptical Dichroism Spectrometer for Cancer Detection

Introduction: Biomolecules are distinct and can be characterized due to their structural differences. Current use instruments for biomolecular structure analysis include X-ray diffraction (XRD), circular dichroism (CD), or nuclear magnetic resonance (NMR) among others. These instruments are in use due to their high resolution and ability to differentiate minor changes in biomolecular structures. However, this comes at a higher cost and the requirements for more caution as these instruments include many delicate parts. CD spectrometry uses circularly polarized light to characterize biomolecules, as well as including laser, photo elastic modulator (PEM), and sometimes cooled photodetectors to increase sensitivity. This limits the available usage of CD spectrometer to lab or clinical settings only due to its bulky size, as well as the need for trained and skilled technicians. To combat this issue, the development of an elliptical dichroism (ED) spectrometer is proposed which uses lower cost components, as well as being markedly smaller, which allows for its integration into more settings such as those areas where accessibility is an issue. However, due to the usage of lower cost components, sensitivity is an issue but is resolvable using components such as gain amplified photodetector. The use of elliptically polarized light also reduces optical components needed and is achieved by use of linear polarized followed by a rotating waveplate to generate elliptically polarized light. Furthermore, these optical components in use for the ED spectrometer are commercially available, lower cost, and are easily accessible allowing for ease of maintenance and repairs if need be.

Materials and

Methods: Instead of a high-powered laser source as seen in CD spectrometry, a broadband UV deuterium lamp was used, followed by a collimator to produce focused light, a linear polarizer to produce linearly polarized light, and a quarter waveplate mounted in a motorized rotator to generate elliptically polarized light. This elliptically polarized light then passes through a cuvette sample holder followed by a GaP gain adjustable amplified photodetector. The optical setup is encompassed inside of a cage offering protection against UV and ozone, as well as heat dissipation with use of vacuum hose connected to the cage. Control of motor for rotating waveplate and photodetector for absorbance readout are done through LabVIEW custom program. Samples of proteins and amino acids used were prepared at 1mg/mL concentrations at a sample volume of 200uL. Samples and solvent blanks were run in repeats of 5 to reduce variability in measurements, and data analyzed using Origin Pro software. Jasco J-815 CD spectrometer was used for validation of ED as well as simulations based on Jones vector and electronic circular dichroism (ECD) using Gaussian09 program package. For cancer diagnostic potential, pancreatic cell lines HPNE and MIA PaCa-2 were obtained from American Type Culture Collection (ATCC). For cancer cell differentiation at the extracellular vesicle (EV) level, EV isolation was performed by ultra-centrifugation.

Results, Conclusions, and Discussions: In AS mode of the ED spectrometer, a full 360° of the rotating waveplate is used which generates a visual representation of biomolecular structure and changes due to exposure to UV. In ED mode, ED value is calculated by the subtraction of absorption values at 45° and 135°, which corresponds to left and right elliptical polarized light, respectively. Simulations for ED spectrometer’s generation of elliptically polarized light were performed using a method based on Jones calculus. Graphing using Poincare sphere shows symmetric profiling of the UV lamp distribution over the range of angles θ = 360°, showing that elliptical polarization at θ = 45° and 135° are symmetric but opposite directions (left and right elliptically polarized light). For validation of the ED spectrometer for its use in analyzing protein structure, BSA and chymotrypsin and avidin were used since these proteins have known secondary structural features. Namely, BSA exhibits α-helix-rich features while chymotrypsin and avidin exhibit β-sheet-rich features. Measurements from the ED spectrometer of these protein samples was compared with CD spectrometer and results found that ED measurements had more variation in BSA values compared to chymotrypsin and avidin suggesting α-helix instability to UV. To analyze ED spectrometer’s use for chirality analysis, AS mode of ED spectrometer was utilized as it provides a graphical representation of the symmetricity and conformational changes of a molecule over time exposure to UV. Additionally, since these conformational changes may cause different variations over time, ED value will yield different values between two different molecules. Compared with time-dependent density functional theory (TD-DFT) and ECD simulation where bond rotation strength is analyzed, ED may offer better insight into experimental methods, something that existing theory may lack thereof. For use in cancer detection of biological samples, cells at lysate and EV level were performed and AUCs compared to standard CD spectrometer. Non-tumorous HPNE and tumorous MIA PaCa-2 had significant differences at both levels in AS graphical representation as well as ED values.

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