Proteins and peptides play a predominant role in biochemical reactions of living cells. In these complex environments, not only the constitution of the molecules but also their three-dimensional configuration defines their functionality. This so-called secondary structure of proteins is crucial for understanding their function in living matter. Misfolding, for example, is suspected as the cause of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Ultimately, it is necessary to study a single protein and its folding dynamics. Here, we report a first step in this direction, namely ultrasensitive detection and discrimination of in vitro polypeptide folding and unfolding processes using resonant plasmonic nanoantennas for surface-enhanced vibrational spectroscopy. We utilize poly-l-lysine as a model system which has been functionalized on the gold surface. By in vitro infrared spectroscopy of a single molecular monolayer at the amide I vibrations we directly monitor the reversible conformational changes between α-helix and β-sheet states induced by controlled external chemical stimuli. Our scheme in combination with advanced positioning of the peptides and proteins and more brilliant light sources is highly promising for ultrasensitive in vitro studies down to the single protein level.
Monosaccharides, which include the simple sugars such as glucose and fructose, are among the most important carbohydrates in the human diet. Certain chronic diseases, e.g., diabetes mellitus, are associated with anomalous glucose blood levels. Detecting and measuring the levels of monosaccharides in vivo or in aqueous solutions is thus of the utmost importance in life science, health, and point-of-care applications. Noninvasive sensing would avoid problems such as pain and potential infection hazards. Here, with the help of surface enhanced infrared absorption (SEIRA) spectroscopy, we demonstrate the reliable optical detection in the mid-infrared spectral range of pure glucose and fructose solutions as well as mixtures of both in aqueous solution. We utilize a reflection flow cell geometry with physiologically relevant concentrations as small as 10 g/L. As significant improvement over the standard baseline correction employed in SEIRA applications, we utilize principal component analysis (PCA) as machine learning algorithm, which is ideally suited for the extraction of vibrational data. We anticipate our results as important step in biosensing applications that will stimulate efforts to further improve the employed SEIRA substrates, the noise level of the spectroscopic light source, as well as the flow cell environment en route to significantly higher sensitivities and quantitative analysis, even in tear drops.
Infrared vibrational spectroscopy is a powerful tool for the identification of changes of the secondary protein structure, which are associated with many human diseases, such as Alzheimer's or Parkinson's disease. In order to obtain deeper insight into the mechanisms that lead to such changes, it is important to investigate the protein conformation on minimum sample concentrations and volumes, ideally on the single-protein level, and at short measurement times. For this purpose, surface-enhanced infrared absorption in combination with Fourier-transform infrared (FTIR) micro-spectroscopy is a highly suitable technique, but is usually limited to large numbers of proteins and long integration times due to the low brilliance of the standard thermal infrared light sources, such as Globar light sources. Here, we push this inherent limit down by using a highly brilliant, broadband mid-IR laser in combination with a standard FTIR microscope. Thus, detecting the secondary protein structure in a sample volume that is ∼100 times smaller than previously demonstrated becomes feasible with our tabletop FTIR system. We utilize polypeptides as a model system, which we functionalize on a single resonant gold nanoantenna, and demonstrate the capabilities of our ultrasensitive system to detect the protein conformation in a living environment within 10 min, without the necessity of frequency tuning the mid-IR laser or any data postprocessing. For comparison, measurements conducted with a synchrotron light source were also performed, which support the results obtained with the laser source. We believe that with further advances it will be possible to scale the process to the ultimate limit of a few or even single proteins and observe the conformational behavior of a few or individual entities in an aqueous environment.
Proteins perform a variety of essential functions in living cells and thus are of critical interest for drug delivery as well as disease biomarkers. The different functions are derived from a hugely diverse set of structures, fueling interest in their conformational states. Surface-enhanced infrared absorption spectroscopy has been utilized to detect and discriminate protein monomers. As an important step forward, we are investigating collagen peptides consisting of a triple helix. While they constitute the main structural building blocks in many complex proteins, they are also a perfect model system for the complex proteins relevant in biological systems. Their complex spectroscopic information as well as the overall small size present a significant challenge for their detection and discrimination. Using resonant plasmonic nanoslits, which are known to show larger specificity compared to nanoantennas, we overcome this challenge. We perform in vitro surface-enhanced absorption spectroscopy studies and track the conformational changes of these collagen peptides under two different external stimuli, which are temperature and chemical surroundings. Modeling the coupling between the amide I vibrational modes and the plasmonic resonance, we can extract the conformational state of the collages and thus monitor the folding and unfolding dynamics of even a single monolayer. This leads to new prospects in studies of single layers of proteins and their folding behavior in minute amounts in a living environment.
Micro-Fourier-transform infrared (FTIR) spectroscopy is a widespread technique that enables broadband measurements of infrared active molecular vibrations at high sensitivity. SiC globars are often applied as light sources in tabletop systems, typically covering a spectral range from about 1 to 20 µm (10 000 -500 cm 1 ) in FTIR spectrometers. However, measuring sample areas below 40x40 µm 2 requires very long integration times due to their inherently low brilliance. This hampers the detection of ultrasmall samples, such as minute amounts of molecules or single nanoparticles. In this publication we extend the current limits of FTIR spectroscopy in terms of measurable sample areas, detection limit and speed by utilizing a broadband, tabletop laser system with MHz repetition rate and femtosecond pulse duration that covers the spectral region between 1250 -7520 cm 1 (1.33 -8 µm). We demonstrate mapping of a 150x150 µm 2 sample of 100 nm thick molecule layers at 1430 cm 1 (7 µm) with 10x10 µm 2 spatial resolution and a scan speed of 3.5 µm/sec. Compared to a similar globar measurement an order of magnitude lower noise is achieved, due to an excellent long-term wavelength and power stability, as well as an orders of magnitude higher brilliance.
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