Noninvasive Monitoring of Glucose Using Near-Infrared Reflection Spectroscopy of Skin-Constraints and Effective Novel Strategy in Multivariate Calibration.

For many years, successful noninvasive blood glucose monitoring assays have been announced, among which near-infrared (NIR) spectroscopy of skin is a promising analytical method. Owing to the tiny absorption bands of the glucose buried among a dominating variable spectral background, multivariate calibration is required to achieve applicability for blood glucose self-monitoring. The most useful spectral range with important analyte fingerprint signatures is the NIR spectral interval containing combination and overtone vibration band regions. A strategy called science-based calibration (SBC) has been developed that relies on a priori information of the glucose signal ("response spectrum") and the spectral noise, i.e., estimates of the variance of a sample population with negligible glucose dynamics. For the SBC method using transcutaneous reflection skin spectra, the response spectrum requires scaling due to the wavelength-dependent photon penetration depth, as obtained by Monte Carlo simulations of photon migration based on estimates of optical tissue constants. Results for tissue glucose concentrations are presented using lip NIR-spectra of a type-1 diabetic subject recorded under modified oral glucose tolerance test (OGTT) conditions. The results from the SBC method are extremely promising, as statistical calibrations show limitations under the conditions of ill-posed equation systems as experienced for tissue measurements. The temporal profile differences between the glucose concentration in blood and skin tissue were discussed in detail but needed to be further evaluated.

Systematic stability testing of insulins as representative biopharmaceuticals using ATR FTIR-spectroscopy with focus on quality assurance.

SIGNIFICANCE: Bioactive proteins represent the most important component class in biopharmaceutical products for therapeutic applications. Their production is most often biotechnologically realized by genetically engineered microorganisms. For the quality assurance of insulins as representatives of life-saving pharmaceuticals, analytical methods are required that allow more than total protein quantification in vials or batches. Chemical and physical factors such as unstable temperatures or shear rate exposure under storage can lead to misfolding, nucleation, and subsequent fibril forming of the insulins. The assumption is valid that these processes go parallel with a decrease in bioactivity. AIM: Infrared (IR) spectroscopy has been successfully utilized for secondary structure analysis in cases of protein misfolding and fibril formation. APPROACH: A reliable method for the quantification of the secondary structure changes has been developed using insulin dry-film Fourier-transform IR spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution, and principal component analysis. RESULTS: A systematic study of insulin spectra was carried out on model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration. Insulin specimens were stored at different temperatures, i.e., 0°C, 20°C, and 37°C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of hormone secondary structure changes, which are supposed to be negatively correlated with insulin bioactivity. CONCLUSIONS: It could be shown that IR-ATR spectroscopy offers a fast and reliable analytical method for the determination of secondary structural changes within insulin molecules, as available in pharmaceutical insulin formulations and therefore challenges internationally established measurement techniques for quality control regarding time, costs, and effort of analysis.

Quality Assurance of Commercial Insulin Formulations: Novel Assay Using Infrared Spectroscopy.

BACKGROUND: For insulins in commercial formulations, degradation can be observed within the certified shelf life when not stored at recommended conditions. Elevated temperatures and exposure to shear forces can cause changes in the secondary structure of the hormone, leading to a decrease in pharmaceutical potency. International pharmacopoeia recommendations for insulin quality monitoring assays mainly rely on liquid chromatography methods. These methods are unable to distinguish between active and inactive forms, both of which may exist in pharmaceutical insulins exposed to stress conditions. METHOD: Infrared attenuated total reflection spectroscopy has been used for the analysis of insulin dry film preparations using affordable instrumentation. This method can be applied to either formulated insulin specimens or pure insulins obtained by ultrafiltration. Such samples have been stored under different temperatures (0°C, 20°C, and 37°C), and degradation processes have been monitored up to a period of a few months. RESULTS: By analyzing specific shifts of absorption bands in the infrared spectra, which are sensitive to the protein secondary structure, even small structural changes in the hormone become evident. Another option is amide I band deconvolution into individual bands, which can be attributed to secondary structure subunits that are part of the insulin tertiary structure. CONCLUSION: A novel and innovative method based on infrared attenuated total reflection spectroscopy of insulin dry films is a promising analytical tool for quantifying the degree of insulin degradation, as it provides information on indicating a decrease in biological potency. The established methods for insulin potency assays require animal testing or clamp experiments on people with diabetes.

Evaluation of Opportunities and Limitations of Mid-Infrared Skin Spectroscopy for Noninvasive Blood Glucose Monitoring.

BACKGROUND: A wide range of optical techniques has recently been presented for the development of noninvasive methods for blood glucose sensing based on multivariate skin spectrum analysis, and most recent studies are reviewed in short by us. The vibrational spectral fingerprints of glucose, as especially found in the mid-infrared or Raman spectrum, have been suggested for achieving largest selectivity for the development of noninvasive blood glucose methods. METHODS: Here, the different aspects on integral skin measurements are presented, which are much dependent on the absorption characteristics of water as the main skin constituent. In particular, different mid-infrared measurement techniques as realized recently are discussed. The limitations of the use of the attenuated total reflection technique in particular are elaborated, and confounding skin or saliva spectral features are illustrated and discussed in the light of recently published works, claiming that the attenuated total reflection technique can be utilized for noninvasive measurements. RESULTS: It will be shown that the penetration depth of the infrared radiation with wavelengths around 10 µm is the essential parameter, which can be modulated by different measurement techniques as with photothermal or diffuse reflection. However, the law of physics is limiting the option of using the attenuated total reflection technique with waveguides from diamond or similar optical materials. CONCLUSIONS: There are confounding features from mucosa, stratum corneum, or saliva, which have been misinterpreted for glucose measurements. Results of an earlier study with multivariate evaluation based on glucose fingerprint features are again referred to as a negative experimental proof.

FT-IR versus EC-QCL spectroscopy for biopharmaceutical quality assessment with focus on insulin-total protein assay and secondary structure analysis using attenuated total reflection.

For the quality control of biopharmaceutical products, which contain proteins as the most important active ingredients, shelf life may be limited due to inappropriate storage conditions or mechanical stress. For insulins as representatives of life-saving pharmaceuticals, analytical methods are needed, which are providing additional information than obtained by assays for total protein quantification. Despite sophisticated formulations, the chemical stability may be challenged by temperatures deviating from recommended conditions or shear rate exposure under storage, leading to misfolding, nucleation, and subsequent fibril formation, accompanied by a decrease in bioactivity. A reliable method for insulin quantification and determination of secondary structure changes has been developed by attenuated total reflection (ATR) Fourier-transform infrared spectroscopy of insulin formulations by a silver halide fiber-coupled diamond probe with subsequent dry-film preparation. A special emphasis has been placed on the protein amide I band evaluation, for which spectral band analysis provides unique information on secondary structure fractions for intact and misfolded insulins. Quantitative measurements are possible down to concentrations of less than 0.5 mg/ml, whereas the dry-film preparation delivers high signal-to-noise ratios due to the prior water evaporation, thus allowing a reliable determination of secondary structure information. Graphical abstract.

Non-invasive monitoring of blood glucose using optical methods for skin spectroscopy-opportunities and recent advances.

Diabetes mellitus is a widespread disease with greatly rising patient numbers expected in the future, not only for industrialized countries but also for regions in the developing world. There is a need for efficient therapy, which can be via self-monitoring of blood glucose levels to provide tight glycemic control for reducing the risks of severe health complications. Advancements in diabetes technology can nowadays offer different sensor approaches, even for continuous blood glucose monitoring. Non-invasive blood glucose assays have been promised for many years and various vibrational spectroscopy-based methods of the skin are candidates for achieving this goal. Due to the small spectral signatures of the glucose hidden among a largely variable background, the largest signal-to-noise ratios and multivariate calibration are essential to provide the method applicability for self-monitoring of blood glucose. Besides multiparameter approaches, recently presented devices based on photoplethysmography with wavelengths in the visible and near-infrared range are evaluated for their potential of providing reliable blood glucose concentration predictions. Graphical abstract ᅟ.

Noninvasive Monitoring of Blood Glucose Using Color-Coded Photoplethysmographic Images of the Illuminated Fingertip Within the Visible and Near-Infrared Range: Opportunities and Questions.

Noninvasive blood glucose assays have been promised for many years and various molecular spectroscopy-based methods of skin are candidates for achieving this goal. Due to the small spectral signatures of the glucose used for direct physical detection, moreover hidden among a largely variable background, broad spectral intervals are usually required to provide the mandatory analytical selectivity, but no such device has so far reached the accuracy that is required for self-monitoring of blood glucose (SMBG). A recently presented device as described in this journal, based on photoplethysmographic fingertip images for measuring glucose in a nonspecific indirect manner, is especially evaluated for providing reliable blood glucose concentration predictions.

It's in your blood: spectral biomarker candidates for urinary bladder cancer from automated FTIR spectroscopy.

Blood samples of urinary bladder cancer (UBC) patients and patients with urinary tract infection were analysed with advanced automated high throughput Fourier transform infrared (HT-FTIR)-spectroscopy. Thin dried film samples were robotically prepared on multi-well titer plates (MTP) for absorbance measurements in transmission mode. Within the absorbance, 1st and 2nd derivative spectra of serum and two plasma preparations, discriminative patterns were identified and validated using bioinformatic tools. The optimal spectral resolution for data acquisition was determined. An accurate discrimination of the patient groups was achieved with three different independent spectral variable sets. The HT-FTIR blood test may support future clinical diagnostics.

FTIR spectroscopy of biofluids revisited: an automated approach to spectral biomarker identification.

The extraction of disease specific information from Fourier transform infrared (FTIR) spectra of human body fluids demands the highest standards of accuracy and reproducibility of measurements because the expected spectral differences between healthy and diseased subjects are very small in relation to a large background absorbance of the whole sample. Here, we demonstrate that with the increased sensitivity of modern FTIR spectrometers, automatisation of sample preparation and modern bioinformatics, it is possible to identify and validate spectral biomarker candidates for distinguishing between urinary bladder cancer (UBC) and inflammation in suspected bladder cancer patients. The current dataset contains spectra of blood serum and plasma samples of 135 patients. All patients underwent cytology and pathological biopsy characterization to distinguish between patients without UBC (46) and confirmed UBC cases (89). A minimally invasive blood test could spare control patients a repeated cystoscopy including a transurethral biopsy, and three-day stationary hospitalisation. Blood serum, EDTA and citrate plasma were collected from each patient and processed following predefined strict standard operating procedures. Highly reproducible dry films were obtained by spotting sub-nanoliter biofluid droplets in defined patterns, which were compared and optimized. Particular attention was paid to the automatisation of sample preparation and spectral preprocessing to exclude errors by manual handling. Spectral biomarker candidates were identified from absorbance spectra and their 1(st) and 2(nd) derivative spectra using an advanced Random Forest (RF) approach. It turned out that the 2(nd) derivative spectra were most useful for classification. Repeat validation on 21% of the dataset not included in predictor training with Linear Discriminant Analysis (LDA) classifiers and Random Forests (RFs) yielded a sensitivity of 93 ± 10% and a specificity of 46 ± 18% for bladder cancer. The low specificity can be most likely attributed to the unbalanced and small number of control samples. Using this approach, spectral biomarker candidates in blood-derived biofluids were identified, which allow us to distinguish between cancer and inflammation, but the observed differences were tiny. Obviously, a much larger sample number has to be investigated to reliably validate such candidates.

A stepwise approach toward closed-loop blood glucose control for intensive care unit patients: results from a feasibility study in type 1 diabetic subjects using vascular microdialysis with infrared spectrometry and a model predictive control algorithm.

BACKGROUND: Glycemic control can reduce the mortality and morbidity of intensive care patients. The CLINICIP (closed-loop insulin infusion for critically ill patients) project aimed to develop a closed-loop control system for this patient group. Following a stepwise approach, we combined three independently tested subparts to form a semiautomatic closed-loop system and evaluated it with respect to safety and performance aspects by testing it in subjects with type 1 diabetes mellitus (T1DM) in a first feasibility trial. METHODS: Vascular microdialysis, a multianalyte infrared spectroscopic glucose sensor, and a standard insulin infusion pump controlled by an adaptive model predictive control (MPC) algorithm were combined to form a closed-loop device, which was evaluated in four T1DM subjects during 30-hour feasibility studies. The aim was to maintain blood glucose concentration in the target range between 80 and 110 mg/dl. RESULTS: Mean plasma glucose concentration was 110.5 ± 29.7 mg/dl. The MPC managed to establish normoglycemia within 105 ± 78 minutes after trial start and managed to maintain glucose concentration within the target range for 47% of the time. The hyperglycemic index averaged to 11.9 ± 5.3 mg/dl. CONCLUSION: Data of the feasibility trial illustrate the device being effective in controlling glycemia in T1DM subjects. However, the monitoring part of the loop must be improved with respect to accuracy and precision before testing the system in the target population.

Transmission infrared spectroscopy of whole blood--complications for quantitative analysis from leucocyte adhesion during continuous monitoring.

Infrared spectroscopy has been applied to analyse glucose and cellular components in whole blood with the aim of developing an online clinical diagnostic and monitoring modality. Leucocyte adsorption onto the CaF(2) windows was observed over a period of several hours under continuous blood flow using a transmission cell of 30 mum path length. This build-up of cellular material on the windows is responsible for diminishing the sample path length under the flow conditions chosen. The adsorption dynamics have been characterised and their impact on glucose monitoring is reported. For short-term monitoring (<2 hours) a standard error of prediction of 11 mg/dL with human citrated blood samples from three different subjects was achieved. Furthermore, the leucocyte build-up was also reported for porcine EDTA blood monitoring. Consequences and testing opportunities with regard to the first stages in the immune cell reaction to the exposure of body-foreign materials to anticoagulated whole blood are discussed.

Recent applications of near-infrared spectroscopy in cancer diagnosis and therapy.

In recent years, near-infrared spectroscopy (NIRS) has gained importance for non-invasive or minimally invasive diagnostic applications in cancer. This technology is based on differences of endogenous chromophores between cancer and normal tissues using either oxy-haemoglobin or deoxy-haemoglobin, lipid or water bands, or a combination of two or more of these as diagnostic markers. These marker bands provide a basis for the diagnosis and therapy monitoring of several cancers. Various applications also use advances in NIR fluorescence spectroscopy which is based on exogenous contrast-enhancing agents. In this review the literature published during the last seven years has been assessed. It will provide an overview on the importance of the NIRS tools in cancer pathology, and in the near future it is envisaged to play a crucial role in cancer diagnosis, treatment decisions, and defining therapeutic drug levels.

Bedside monitoring of subcutaneous interstitial glucose in healthy individuals using microdialysis and infrared spectrometry.

An IR-spectroscopy-based bedside device, coupled to a subcutaneously implanted microdialysis probe, is developed for quasicontinuous glucose monitoring with intermittent readouts at 10-min intervals, avoiding any sensor recalibration under long-term operation. The simultaneous estimation of the microdialysis recovery rate is possible using an acetate containing perfusate and determining its losses across the dialysis membrane. Measurements are carried out on four subjects, with experiments lasting either 8 or 28 h, respectively. Using the spectral interval data either from 1180 to 950 or 1560 to 1000 cm(-1), standard errors of prediction (SEPs) between 0.13 and 0.28 mM are achieved using multivariate calibration with partial least-squares (PLS) or classical least-squares (CLS) calibration models, respectively. The transfer of a PLS calibration model using the spectral and reference concentration data of the dialysates from the three 8-h-long experiments to a 28-h monitoring episode with another healthy subject is tested. Including microdialysis recovery for the determination of the interstitial glucose concentrations, an SEP of 0.24 mM is obtained versus whole blood glucose values. The option to determine other metabolites such as urea or lactate offers the possibility to develop a calibration- and reagent-free point-of-care analyzer.

Recent progress in analytical instrumentation for glycemic control in diabetic and critically ill patients.

Implementing strict glycemic control can reduce the risk of serious complications in both diabetic and critically ill patients. For this reason, many different analytical, mainly electrochemical and optical sensor approaches for glucose measurements have been developed. Self-monitoring of blood glucose (SMBG) has been recognised as being an indispensable tool for intensive diabetes therapy. Recent progress in analytical instrumentation, allowing submicroliter samples of blood, alternative site testing, reduced test time, autocalibration, and improved precision, is comprehensively described in this review. Continuous blood glucose monitoring techniques and insulin infusion strategies, developmental steps towards the realization of the dream of an artificial pancreas under closed loop control, are presented. Progress in glucose sensing and glycemic control for both patient groups is discussed by assessing recent published literature (up to 2006). The state-of-the-art and trends in analytical techniques (either episodic, intermittent or continuous, minimal-invasive, or noninvasive) detailed in this review will provide researchers, health professionals and the diabetic community with a comprehensive overview of the potential of next-generation instrumentation suited to either short- and long-term implantation or ex vivo measurement in combination with appropriate body interfaces such as microdialysis catheters.

CH-overtone regions as diagnostic markers for near-infrared spectroscopic diagnosis of primary cancers in human pancreas and colorectal tissue.

We have investigated the application of near-infrared spectroscopy for detection of human primary pancreatic and colorectal cancers. Spectra from cancerous and normal tissue were collected from a total of 37 surgically resected pancreatic and colorectal patient tissue specimens using a fibre-optic probe. Major spectral differences were observed in the CH-stretching first (6,000-5,400 cm(-1)) and second overtone (9,000-7,900 cm(-1)) regions. By use of artificial neural networks, linear discriminant analysis, and cluster analysis as pattern-recognition methods the spectra were classified into cancerous and normal tissue groups with accuracy up to 89%. We also explored differences between the spectra obtained from colorectal and pancreatic tissue. Spectral data from cancerous and normal tissue were classified organ-specifically into four groups with accuracy between 80 and 83%. Our results indicate that CH-overtone regions, besides serving as diagnostic markers for NIR spectroscopic diagnosis of primary human pancreas and colorectal cancers, are also useful for elucidating differences between the spectra obtained from colorectal and pancreatic cancerous tissue.

Infrared transmission spectrometry for the determination of urea in microliter sample volumes of blood plasma dialysates.

Application of mid-infrared spectroscopy for the determination of urea in blood plasma dialysates of microliter sample volumes using a transmission microcell was investigated. Infrared spectra of the dialysates of plasma samples collected from 75 different patients using CMA 60 microdialysis catheters were evaluated with multivariate partial least squares regression. Using the absorbance spectral data from 1520-1420 cm(-1) and 1220-1120 cm(-1), a minimum standard error of prediction (SEP) of 0.88 mg/dL (0.14 mM) was achieved with spectral variable selection. Our findings suggest the feasibility of developing a mid-infrared sensor in combination with micro-fluidics for on-line monitoring of urea in patients undergoing dialysis treatment.

Spectral variable selection for partial least squares calibration applied to authentication and quantification of extra virgin olive oils using Fourier transform Raman spectroscopy.

The limits of quantitative multivariate assays for the analysis of extra virgin olive oil samples from various Greek sites adulterated by sunflower oil have been evaluated based on their Fourier transform (FT) Raman spectra. Different strategies for wavelength selection were tested for calculating optimal partial least squares (PLS) models. Compared to the full spectrum methods previously applied, the optimum standard error of prediction (SEP) for the sunflower oil concentrations in spiked olive oil samples could be significantly reduced. One efficient approach (PMMS, pair-wise minima and maxima selection) used a special variable selection strategy based on a pair-wise consideration of significant respective minima and maxima of PLS regression vectors, calculated for broad spectral intervals and a low number of PLS factors. PMMS provided robust calibration models with a small number of variables. On the other hand, the Tabu search strategy recently published (search process guided by restrictions leading to Tabu list) achieved lower SEP values but at the cost of extensive computing time when searching for a global minimum and less robust calibration models. Robustness was tested by using packages of ten and twenty randomly selected samples within cross-validation for calculating independent prediction values. The best SEP values for a one year's harvest with a total number of 66 Cretian samples were obtained by such spectral variable optimized PLS calibration models using leave-20-out cross-validation (values between 0.5 and 0.7% by weight). For the more complex population of olive oil samples from all over Greece (total number of 92 samples), results were between 0.7 and 0.9% by weight with a cross-validation sample package size of 20. Notably, the calibration method with Tabu variable selection has been shown to be a valid chemometric approach by which a single model can be applied with a low SEP of 1.4% for olive oil samples across three different harvest years.

Nanoliter serum sample analysis by mid-infrared spectroscopy for minimally invasive blood-glucose monitoring.

The aim of this study was to demonstrate that mid-infrared spectroscopy is able to quantify glucose in a serum matrix with sample volumes well below 1 muL. For this, we applied mid-infrared attenuated total reflectance (ATR) or transmission-based spectroscopic methods to glucose quantification in microsamples of dry-film sera, either undiluted or diluted 10 times in distilled water. The sample series spanned physiological glucose concentrations between 50 and 600 mg/dL and volumes of 80, 8, and 1 nL. Calibration was carried out using multivariate partial least-squares (PLS) modeling with spectral data between 1180 and 940 cm(-1). Best performance was achieved in the ATR experiments. For raw ATR spectra, the optimum standard error of prediction (SEP) of 13.3 mg/dL was obtained for the 8 nL sample series with subsequent 10-fold dilution. With respect to the coefficient of variation of the glucose assay, CV(pred), we obtained a value of 3% for the 80 nL volume samples with spectral preprocessing using matrix protein absorption bands as an internal standard, 4% for the 8 nL samples, and 6% for the 1 nL samples with raw data. Spectral standardization resulted in significant improvement, especially for the 80 nL volume sample series. By contrast, the accuracy of the glucose assay for the 1 nL sample volume series could not be improved either by internal standardization or by considering the dry film areas for normalization, which we attribute to varying topographies of the dry films.

Glucose quantification in dried-down nanoliter samples using mid-infrared attenuated total reflection spectroscopy.

The aim of this study was to determine the feasibility of minimally invasive glucose concentration measurement of a body fluid within the physiologically important range below 100 nL with a number of samples such as interstitial fluid, plasma, or whole blood using mid-infrared spectroscopy, but starting with preliminary measurements on samples of simple aqueous glucose solutions. The Fourier transform infrared spectrometer was equipped with a Golden Gate single reflection diamond attenuated total reflection (ATR) accessory and a room-temperature pyroelectric detector. As the necessary detection limits can be achieved only for dried samples within the spectrometric conditions realized by a commercial instrument, the work focused on the optimization of such ATR measurements. We achieved quantification of samples with volumes as low as 7 nL between 10 and 600 mg/dL. The standard error of prediction (SEP) for the concentration range 10-100 mg/dL is 3.2 mg/dL with full interval data between 1180 and 940 cm(-1). The performance of the prediction is given by a coefficient of variation of prediction (CV(pred) ) of 6.2%. When all samples within the whole concentration range are included, the SEP increases to 20.2 mg/dL, and hence the CV(pred) to 10.6% due to a nonlinear signal dependence on glucose concentration. A detection limit for glucose of 0.7 ng with a signal-to-noise ratio of 10 was obtained.