Optical Screening and Classification of Drug Binding to Proteins in Human Blood Serum.

Protein-drug interactions in the human bloodstream are important factors in applications ranging from drug design, where protein binding influences efficacy and dose delivery, to biomedical diagnostics, where rapid, quantitative measurements could guide optimized treatment regimes. Current measurement approaches use multistep assays, which probe the protein-bound drug fraction indirectly and do not provide fundamental structural or dynamic information about the in vivo protein-drug interaction. We demonstrate that ultrafast 2D-IR spectroscopy can overcome these issues by providing a direct, label-free optical measurement of protein-drug binding in blood serum samples. Four commonly prescribed drugs, known to bind to human serum albumin (HSA), were added to pooled human serum at physiologically relevant concentrations. In each case, spectral changes to the amide I band of the serum sample were observed, consistent with binding to HSA, but were distinct for each of the four drugs. A machine-learning-based classification of the serum samples achieved a total cross-validation prediction accuracy of 92% when differentiating serum-only samples from those with a drug present. Identification on a per-drug basis achieved correct drug identification in 75% of cases. These unique spectroscopic signatures of the drug-protein interaction thus enable the detection and differentiation of drug containing samples and give structural insight into the binding process as well as quantitative information on protein-drug binding. Using currently available instrumentation, the 2D-IR data acquisition required just 1 min and 10 µL of serum per sample, and so these results pave the way to fast, specific, and quantitative measurements of protein-drug binding in vivo with potentially invaluable applications for the development of novel therapies and personalized medicine.

2D-IR spectroscopy of proteins in H2O-A Perspective.

The form of the amide I infrared absorption band provides a sensitive probe of the secondary structure and dynamics of proteins in the solution phase. However, the frequency coincidence of the amide I band with the bending vibrational mode of H2O has necessitated the widespread use of deuterated solvents. Recently, it has been demonstrated that ultrafast 2D-IR spectroscopy allows the detection of the protein amide I band in H2O-based fluids, meaning that IR methods can now be applied to study proteins in physiologically relevant solvents. In this perspective, we describe the basis of the 2D-IR method for observing the protein amide I band in H2O and show how this development has the potential to impact areas ranging from our fundamental appreciation of protein structural dynamics to new applications for 2D-IR spectroscopy in the analytical and biomedical sciences. In addition, we discuss how the spectral response of water, rather than being a hindrance, now provides a basis for new approaches to data pre-processing, standardization of 2D-IR data collection, and signal quantification. Ultimately, we visualize a direction of travel toward the creation of 2D-IR spectral libraries that can be linked to advanced computational methods for use in high-throughput protein screening and disease diagnosis.

Measuring proteins in H2O using 2D-IR spectroscopy: pre-processing steps and applications toward a protein library.

The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in H2O rather than D2O-based solvents by evading the interfering water signals has enabled in vivo studies of proteins under physiological conditions and in biofluids. Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will, however, require comparisons between multiple datasets, necessitating control of data collection protocols to minimize measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalization strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement. These advances show progress toward the robust data handling strategies that will be necessary for future applications of 2D-IR to pharmaceutical or biomedical problems.

Detection of paracetamol binding to albumin in blood serum using 2D-IR spectroscopy.

Binding of drugs to blood serum proteins can influence both therapeutic efficacy and toxicity. The ability to measure the concentrations of protein-bound drug molecules quickly and with limited sample preparation could therefore have considerable benefits in biomedical and pharmaceutical applications. Vibrational spectroscopies provide data quickly but are hampered by complex, overlapping protein amide I band profiles and water absorption. Here, we show that two-dimensional infrared (2D-IR) spectroscopy can achieve rapid detection and quantification of paracetamol binding to serum albumin in blood serum at physiologically-relevant levels with no additional sample processing. By measuring changes to the amide I band of serum albumin caused by structural and dynamic impacts of paracetamol binding we show that drug concentrations as low as 7 µM can be detected and that the availability of albumin for paracetamol binding is less than 20% in serum samples, allowing identification of paracetamol levels consistent with a patient overdose.

Clinical Spectroscopy: Lost in Translation?

This Focal Point Review paper discusses the developments of biomedical Raman and infrared spectroscopy, and the recent strive towards these technologies being regarded as reliable clinical tools. The promise of vibrational spectroscopy in the field of biomedical science, alongside the development of computational methods for spectral analysis, has driven a plethora of proof-of-concept studies which convey the potential of various spectroscopic approaches. Here we report a brief review of the literature published over the past few decades, with a focus on the current technical, clinical, and economic barriers to translation, namely the limitations of many of the early studies, and the lack of understanding of clinical pathways, health technology assessments, regulatory approval, clinical feasibility, and funding applications. The field of biomedical vibrational spectroscopy must acknowledge and overcome these hurdles in order to achieve clinical efficacy. Current prospects have been overviewed with comment on the advised future direction of spectroscopic technologies, with the aspiration that many of these innovative approaches can ultimately reach the frontier of medical diagnostics and many clinical applications.

Differentiation of bacterial spores via 2D-IR spectroscopy.

Ultrafast 2D-IR spectroscopy is a powerful tool for understanding the spectroscopy and dynamics of biological molecules in the solution phase. A number of recent studies have begun to explore the utility of the information-rich 2D-IR spectra for analytical applications. Here, we report the application of ultrafast 2D-IR spectroscopy for the detection and classification of bacterial spores. 2D-IR spectra of Bacillus atrophaeus and Bacillus thuringiensis spores as dry films on CaF2 windows were obtained. The sporulated nature of the bacteria was confirmed using 2D-IR diagonal and off-diagonal peaks arising from the calcium dipicolinate CaDP·3H2O biomarker for sporulation. Distinctive peaks, in the protein amide I region of the spectrum were used to differentiate the two types of spore. The identified marker modes demonstrate the potential for the use of 2D-IR methods as a direct means of spore classification. We discuss these new results in perspective with the current state of analytical 2D-IR measurements, showing that the potential exists to apply 2D-IR spectroscopy to detect the spores on surfaces and in suspensions as well as in dry films. The results demonstrate how applying 2D-IR screening methodologies to spores would enable the creation of a library of spectra for classification purposes.

Detection of Glycine as a Model Protein in Blood Serum Using 2D-IR Spectroscopy.

Glycine (Gly) is used as a model system to evaluate the ability of ultrafast two-dimensional infrared (2D-IR) spectroscopy to detect and quantify the low-molecular-weight proteinaceous components of blood serum. Combining data acquisition schemes to suppress absorption bands of H2O that overlap with the protein amide I band with analysis of peak patterns appearing in the off-diagonal region of the 2D-IR spectrum allows separation of the Gly spectral signature from that of the dominant protein fraction of serum in a transmission-mode 2D-IR measurement without any sample manipulation, e.g., filtration or drying. 2D-IR spectra of blood serum samples supplemented with varying concentrations of Gly were obtained, and a range of data analysis methods compared, leading to a detection limit of ∼3 mg/mL for Gly. The reported methodology provides a platform for a critical assessment of the sensitivity of 2D-IR for measuring the concentrations of amino acids, peptides, and low-molecular-weight proteins present in serum samples. We conclude that, in the case of several clinically relevant diagnostic molecules and their combinations, the potential exists for 2D-IR to complement IR absorption methods as the benefits of the second frequency dimension offered by 2D-IR spectroscopy outweigh the added technical complexity of the measurement.