We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future.

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the non-enzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and non-glycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision - with a limit of detection that is nearly a factor of 15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multi-component mixtures and potentially in hemolysate and whole blood lysate samples.

We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.

Microcalcifications are a feature of diagnostic significance on a mammogram and a target for stereotactic breast needle biopsy. Here, we report development of a Raman spectroscopy technique to simultaneously identify microcalcification status and diagnose the underlying breast lesion, in real-time, during stereotactic core needle biopsy procedures. Raman spectra were obtained ex vivo from 146 tissue sites from fresh stereotactic breast needle biopsy tissue cores from 33 patients, including 50 normal tissue sites, 77 lesions with microcalcifications, and 19 lesions without microcalcifications, using a compact clinical system. The Raman spectra were modeled based on the breast tissue components and a support vector machine framework was used to develop a single-step diagnostic algorithm to distinguish normal tissue, fibrocystic change (FCC), fibroadenoma (FA) and breast cancer, in the absence and presence of microcalcifications. This algorithm was subjected to leave-one-site-out cross-validation, yielding a positive predictive value, negative predictive value, sensitivity and specificity of 100%, 95.6%, 62.5% and 100% for diagnosis of breast cancer (with or without microcalcifications) and an overall accuracy of 82.2% for classification into specific categories of normal tissue, FCC, FA or breast cancer (with and without microcalcifications). Notably, the majority of breast cancers diagnosed are ductal carcinoma in situ (DCIS), the most common lesion associated with microcalcifications, which could not be diagnosed using previous Raman algorithm(s). Our study demonstrates the potential of Raman spectroscopy to concomitantly detect microcalcifications and diagnose associated lesions, including DCIS, and thus provide real-time feedback to radiologists during such biopsy procedures, reducing non-diagnostic and false negative biopsies.