Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm–1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein–protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.

Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about10 ms with 16 cm−1 resolution), step-scan FTIR spectroscopy reaches a time-resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of non-repetitive reactions, e.g. induced by caged compounds, were limited to the ms time domain. The advent of dual comb quantum cascade laser allows now for a rapid reaction monitoring in the μs time domain. Here we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound induced reactions monitored by FTIR and dual comb spectroscopy, respectively, by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable μs time regime for non-repetitive biological systems including all GTPases and ATPases.

Background The immuno‐infrared‐sensor enables the diagnosis of Alzheimer’s disease by measuring the secondary structure distribution of Amyloid‐beta (Aß) in CSF and blood plasma. For this technique an antibody is essential, which binds to all conformations of the biomarker peptide. We developed different immunization strategies to generate monoclonal Aß‐antibodies (mAb) using wt and APP Knock‐Out (KO) mice. Method KO and wt mice were immunized with different Aß peptides. With an antigen‐specific ELISA antibody‐producing hybridoma cells were screened and selected for performance tests with the immuno‐infrared sensor. Immuno‐infrared‐analyses provided a detailed characterization of the structural selectivity of the antibodies. Result Both KO and wt mice generated multiple Aß specific antibodies, though KO mice yielded 8 times more Aß‐positive antibodies than wt. All antibodies, probably polyclonal, revealed binding to alpha‐helical or disordered monomeric Aß as well as to ß‐sheet enriched Aß (oligomers, fibrils). Additionally, seven of these antibodies differentiated between AD patients and healthy controls in cerebrospinal fluid (CSF) samples. Conclusion With our immunization strategy we were able to generate multiple antibodies for the diagnosis of Alzheimer’s disease with the immuno‐infrared sensor. We generated seven antibodies, which enable the differentiation of AD patients and healthy controls by analyses on CSF. References: [1] Nabers, A., Ollesch J. et al. (2016) J. Biophotonics, 9. 224‐234. [2] Nabers, A. et al. (2018) EMBO Mol. Med. 10, e8763. [3] Nabers, A et al. (2019) Alzheimer’s Dement. Diagnosis, assess. Dis. Monit., 11, 257‐263. [4] Stocker, H et al. (2019), Alzheimer's & Dementia, in press.

Background It is believed, that one of the first events in the progression of Alzheimer’s Disease is the misfolding of the Amyloid Beta (Aβ) Peptide. This event takes place up to 20 years before the clinical onset of the disease. Therefore, biosensors detecting the misfolding of Aβ are especially useful for the early and specific detection of Alzheimer’s Disease. In recent studies, our biosensor based on ATR‐FTIR spectroscopy performed with an overall accuracy of 86% [1,2,3]. This system is robust and reliable but the user input required is very high because each sensor can only be used once. To overcome these issues and increase the automation capacity, a regenerative system is urgently needed. Method To achieve higher throughput of this system, we introduced immunoglobulin binding proteins (Protein A and Protein G) as capture for antibody immobilization. Protein A and Protein G are able to recognize and bind the constant region of different IgG antibodies in a non‐covlant manner. This allows multiple measurements within the same system by performing multiple binding and elution cycles. Result Protein A and Protein G can be attached covalently to the sensor without losing its ability to bind IgG antibodies [4]. The antibody binding and elution is monitored by ATR‐FTIR spectroscopy and validated using fluorescence spectroscopy. It is possible to perform at least 35 cycles of antibody binding and elution over 7 days without loss in diagnostic accuracy. The sensor is still able to extract different biomarkers like Aβ and the Tau Protein from CSF with highly comparable results compared to the recently used system. Conclusion The development of a regenerative immuno‐infrared‐sensor is crucial to reduce the user input and to increase the throughput of this method. Compared to assays where the capture antibody is covalently immobilized, the regenerative system has a factor 6 increased thoughput. References: (1) Nabers, A. et al. (2016) J. Biophotonics, 9. 224‐234. (2) Nabers, A. et al. (2018) EMBO Mol. Med. 10, e8763. (3) Nabers, A et al. (2019) Alzheimer’s Dement. Diagnosis, Assess. Dis. Monit., 11, 257‐263. (4) Budde, B. et al. (2019) ACS Sens. 4, 1851‐1856.