Hemoglobinopathies are the most common genetic disorders caused by a mutation in the genes encoding for one of the globin chains and leading to structural (hemoglobin [Hb] variants) or quantitative defects (thalassemias) in hemoglobin. Early diagnosis and characterization of hemoglobinopathies are essential to avoid severe hematological consequences in the offspring of healthy carriers of a mutation. Despite being extensively studied, hemoglobinopathies continue to provide a diagnostic challenge. Sickle-cell hemoglobin (HbS) is the most common and clinically significant hemoglobin variant among all Hb variants. To overcome the challenge of diagnosing Hb variants, we propose the use of Surface-Enhanced Raman Spectroscopy (SERS). SERS is a powerful label-free tool for providing fingerprint structural information of analyses. It can rapidly generate the spectral signature of samples. This study investigates the structural differences between HbS and normal Hb using gold nanopillar SERS substrates with a leaning effect. The SERS spectra of Hb variants showed subtle spectral differences between HbS and normal Hb located in the valine (975 cm-1) and glutamic acid (1547 cm-1) band, reflecting the amino acid substitution in the HbS β-globin chain. We also automated the identification of HbS and normal Hb with principal component analysis (PCA) combined with support vector machine (SVM) and linear discriminant analysis (LDA) classifiers, leading to an accuracy of 98% and 96%, respectively. This study demonstrated that SERS can provide a fast, highly sensitive, noninvasive, and accurate detection module for the diagnosis of Sickle-cell disease and potentially other hemoglobinopathies.
Raman spectroscopy is a powerful tool for analytical measurements in many applications. Traditional Raman spectroscopic analyses require bulky equipment, considerable time of signal acquisition and manual sampling of substances under test. In this paper, we take a step from bulky and manual consuming laboratory testing towards lab-on-chip (LOC) analyses. We miniaturize the Raman spectroscopic system by combining a free-form reflector based polymer LOC with a customized Raman probe. By using the confocal detection principle, we aim to enhance the detection of the Raman signals from the substance of interest due to the suppression of the background Raman signal from the polymer of the chip. Next to the LOC we miniaturize the external optical components, surrounding the reflector embedding optofluidic chip, and assemble these in a Raman probe. We evaluate the misalignment tolerance of internal optics (LOC) and external optics (Raman probe) by non-sequential ray tracing which shows that off-axis misalignment is around ±400μm and the maximum working distance of our Raman probe is 71mm. Using this probe, the system could be implemented as a portable reader unit containing the external optics, in which a low-cost, robust and mass manufacturable microfluidic LOC containing a freeform reflector is inserted, to enable confocal Raman spectroscopy measurements.
Traditionally, Raman spectroscopy is done in a specialized lab, with considerable requirements in terms of equipment, time and manual sampling of substances of interest. We present the modeling, the design and the fabrication process of a microfluidic device incorporation Raman spectroscopy, from which one enables confocal Raman measurements on-chip. The latter is fabricated using ultra precision diamond tooling and is tested in a proof-of-concept setup, by for example measuring Raman spectra of urea solutions with various concentrations. If one wants to analyze single cells instead of a sample solution, precautions need to be taken. Since Raman scattering is a weak process, the molecular fingerprint of flowing particles would be hard to measure. One method is to stably position the cell under test in the detection area during acquisition of the Raman scattering such that the acquisition time can be increased. Positioning of cells can be done through optical trapping and leads to an enhanced signal-to-noise ratio and thus a more reliable cell identification. Like Raman spectroscopy, optical trapping can also be miniaturized. We present the modeling, design process and fabrication of a mass-manufacturable polymer microfluidic device for dual fiber optical trapping using two counterpropagating singlemode beams. We use a novel fabrication process that consists of a premilling step and ultraprecision diamond tooling for the manufacturing of the molds and double-sided hot embossing for replication, resulting in a robust microfluidic chip for optical trapping. In a proof-of-concept demonstration, we characterize the trapping capabilities of the hot embossed chip.
Raman spectroscopy is a powerful optical and non-destructive technique and a well-known method for analysis purposes, especially to determine the molecular fingerprint of substances. Traditionally, such analyses are done in a specialized lab, with considerable requirements in terms of equipment, time and manual sampling of substances of interest. In this paper we take a step from bulky Raman spectroscopy laboratory analyses towards lab-on-chip (LOC) analyses. We present an optofluidic lab-on-chip for confocal Raman spectroscopy, which can be used for the analysis of liquids. The confocal detection suppresses the unwanted background from the polymer material out of which the chip is fabricated. We design the free-form optical reflector using non-sequential ray-tracing combined with a mathematical code to simulate the Raman scattering behavior of the substance under test. We prototype the device in Polymethyl methacrylate (PMMA) by means of ultraprecision diamond tooling. In a proof-of-concept demonstration, we first show the confocal behavior of our Raman lab-on-chip system by measuring the Raman spectrum of ethanol. In a next step, we compare the Raman spectra measured in our lab-on-chip with spectra measured with a commercial Raman spectrometer. Finally, to calibrate the system we perform Raman measurements on urea solutions with different concentrations. We achieve a detection limit that corresponds to a noise equivalent concentration of 20mM. Apart from strongly reducing the background perturbations, our confocal Raman spectroscopy system has other advantages as well. The reflector design is robust from a mechanical point of view and has the potential for mass-manufacturing using hot embossing or injection molding.
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