Serum is an important candidate in proteomics analysis as it potentially carries key markers on health status and disease progression. However, several important diagnostic markers found in the circulatory proteome and the low-molecular-weight (LMW) peptidome have become analytically challenging due to the high dynamic concentration range of the constituent protein/peptide species in serum. Herein, we propose a novel approach to improve the limit of detection (LoD) of LMW amino acids by combining mid-IR (MIR) and near-IR spectroscopic data using glycine as a model LMW analyte. This is the first example of near-IR spectroscopy applied to elucidate the detection limit of LMW components in serum; moreover, it is the first study of its kind to combine mid-infrared (25–2.5 μm) and near-infrared (2500–800 nm) to detect an analyte in serum. First, we evaluated the prediction model performance individually with MIR (ATR-FTIR) and NIR spectroscopic methods using partial least squares regression (PLS-R) analysis. The LoD was found to be 0.26 mg/mL with ATR spectroscopy and 0.22 mg/mL with NIR spectroscopy. Secondly, we examined the ability of combined spectral regions to enhance the detection limit of serum-based LMW amino acids. Supervised extended wavelength PLS-R resulted in a root mean square error of prediction (RMSEP) value of 0.303 mg/mL and R² value of 0.999 over a concentration range of 0–50 mg/mL for glycine spiked in whole serum. The LoD improved to 0.17 mg/mL from 0.26 mg/mL. Thus, the combination of NIR and mid-IR spectroscopy can improve the limit of detection for an LMW compound in a complex serum matrix.

Malaria is a life-threatening disease caused by a parasite, which can be transmitted to humans through bites of infected female Anopheles mosquitoes. This disease plagues a significant population of the world, necessitating the need for better diagnostic platforms to enhance the detection sensitivity, whilst reducing processing times, sample volumes and cost. A critical step in achieving improved detection is the effective lysis of blood samples. Here, we propose the use of an acoustically actuated microfluidic mixer for enhanced blood cell lysis. Guided by numerical simulations, we experimentally demonstrate that the device is capable of lysing a 20× dilution of isolated red blood cells (RBCs) with an efficiency of ∼95% within 350 ms (0.1 mL). Further, experimental results show that the device can effectively lyse whole blood irrespective of its dilution factor. Compared to the conventional method of using water, this platform is capable of releasing a larger quantity of haemoglobin into plasma, increasing the efficiency without the need for lysis reagents. The lysis efficiency was validated with malaria infected whole blood samples, resulting in an improved sensitivity as compared to the unlysed infected samples. Partial least squares-regression (PLS-R) analysis exhibits cross-validated R² values of 0.959 and 0.98 from unlysed and device lysed spectral datasets, respectively. Critically, as expected, the root mean square error of cross validation (RMSECV) value was significantly reduced in the acoustically lysed datasets (RMSECV of 0.97), indicating the improved quantification of parasitic infections compared to unlysed datasets (RMSECV of 1.48). High lysis efficiency and ultrafast processing of very small sample volumes makes the combined acoustofluidic/spectroscopic approach extremely attractive for point-of-care blood diagnosis, especially for detection of neonatal and congenital malaria in babies, for whom a heel prick is often the only option for blood collection.

The identification of biomarkers from blood plasma is at the heart of many diagnostic tests. These tests often need to be conducted frequently and quickly, but the logistics of sample collection and processing not only delays the test result, but also puts a strain on the healthcare system due to the sheer volume of tests that need to be performed. The advent of microfluidics has made the processing of samples quick and reliable, with little or no skill required on the user’s part. However, while several microfluidic devices have been demonstrated for plasma separation, none of them have validated the chemical integrity of the sample post-process. Here, we present Haemoprocessor: a portable, robust, open-fluidic system that utilizes Travelling Surface Acoustic Waves (TSAW) with the expression of overtones to separate plasma from 20× diluted human blood within a span of 2 min to achieve 98% RBC removal. The plasma and red blood cell separation quality/integrity was validated through Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy and multivariate analyses to ascertain device performance and reproducibility when compared to centrifugation (the prevailing gold-standard for plasma separation). Principal Component Analysis (PCA) showed a remarkable separation of 92.21% between RBCs and plasma components obtained through both centrifugation and Haemoprocessor methods. Moreover, a close association between plasma isolates acquired by both approaches in PCA validated the potential of the proposed system as an eminent cell enrichment and plasma separation platform. Thus, compared to contemporary acoustic devices, this system combines the ease of operation, low sample requirement of an open system, the versatility of a SAW device using harmonics, and portability.

Oxidative treatment of human red blood cells (RBCs) prior to freeze-drying appears to stabilize the RBCs to withstand dried storage at room temperature. To better understand the effects of oxidation and freeze-drying/rehydration on RBC lipids and proteins, single-cell measurements were performed by synchrotron-based Fourier transform infrared (FTIR) microspectroscopy ‘live-cell’ (unfixed) analysis. Lipid and protein spectral data of tert-butyl hydroperoxide (TBHP)-oxidized RBCs (oxRBCs), FDoxRBCs and control (untreated) RBCs were compared using principal component analysis (PCA) and band integration ratios. The oxRBCs and FDoxRBCs samples had similar spectral profiles that were clearly different to control RBCs. Spectral changes in the CH stretching region of oxRBCs and FDoxRBCs indicated the presence of increased saturated and shorter-chain lipids, consistent with lipid peroxidation and stiffening of the RBC membrane compared to control RBCs. The PCA loadings plot for the fingerprint region of control RBCs corresponding to the α-helical structure of hemoglobin, shows that oxRBCs and FDoxRBCs have conformational changes in the protein secondary structure to β-pleated sheets and β-turns. Finally, the freeze-drying process did not appear to compound or induce additional changes. In this context, FDoxRBCs could become a stable source of reagent RBCs for pre-transfusion blood serology testing. The synchrotron FTIR microspectroscopic live-cell protocol provides a powerful analytical tool to characterize and contrast the effects of different treatments on RBC chemical composition at the single cell level.