Quantification and Identification of Microproteinuria using Ultrafiltration and ATR-FTIR Spectroscopy

The presence of low amounts of specific proteins in urine can be an indicator of diagnosis and prognosis of several diseases including renal failure and cancer. Hence, there is an urgent need for Point-of-care (PoC) methods, which can quantify microproteinuria levels (30-300 ppm) and identify the major proteins associated with the microproteinuria. In this study, we coupled ultracentrifugation with attenuated total reflectance-Fourier transform infrared (ATR-FTIR) to identify and quantify proteins in urine at low parts per million levels. The process involves the preconcentration of proteins from 500 μL of urine using an ultrafiltration device. After several washings, the isolated proteins are dried onto the ATR crystal forming a thin film. Imaging studies showed that the absorbance of the protein bands was linear with the amount of mass deposited on the crystal. The methodology was first evaluated with artificial urine spiked with 30-300 ppm of albumin. The calibration showed acceptable linearity (R2 = 0.97) and a limit of detection of 6.7 ppm. Linear relationships were also observed from urine of healthy subjects spiked with microproteinuria concentrations of albumin, immunoglobulin, and hemoglobin, giving a prediction error of the spiked concentration of 23 ppm. When multiple proteins were spiked into the real urine, multivariate analysis was able to decompose the data set into the different proteins, but the multicomponent evaluation was challenging for proteins at low levels. Although the introduction of a preprocessing step reduces the PoC capability of the method, it largely increases its performance, showing great potential as a tool for the diagnosis and prognosis of several illnesses affecting urine proteic compositio

Synchrotron Macro ATR-FTIR Microspectroscopy for High Resolution Chemical Mapping of Single Cells

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called “hybrid” macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 μm and 100 μm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science

Spectropathology for the Next Generation: Quo vadis?

Although the potential of vibrational spectroscopy for biomedical applications has been well demonstrated, translation into clinical practice has been relatively slow. This Editorial assesses the challenges facing the field and the potential way forward. While many technological challenges have been addressed to date, considerable effort is still required to gain acceptance of the techniques among the medical community, standardise protocols, extend to a clinically relevant scale, and ultimately assess the health economics underlying clinical deployment. National and international research networks can contribute much to technology development and standardisation. Ultimately, large-scale funding is required to engage in clinical trials and instrument development