Determination of latent tuberculosis infection from plasma samples via label-free SERS sensors and machine learning.

Effective diagnostic tools for screening of latent tuberculosis infection (LTBI) are lacking. We aim to investigate the performance of LTBI diagnostic approaches using label-free surface-enhanced Raman spectroscopy (SERS). We used 1000 plasma samples from Northeast Thailand. Fifty percent of the samples had tested positive in the interferon-gamma release assay (IGRA) and 50 % negative. The SERS investigations were performed on individually prepared protein specimens using the Raman-mapping technique over a 7 × 7 grid area under measurement conditions that took under 10 min to complete. The machine-learning analysis approaches were optimized for the best diagnostic performance. We found that the SERS sensors provide 81 % accuracy according to train-test split analysis and 75 % for LOOCV analysis from all samples, regardless of the batch-to-batch variation of the sample sets and SERS chip. The accuracy increased to 93 % when the logistic regression model was used to analyze the last three batches of samples, following optimization of the sample collection, SERS chips, and database. We demonstrated that SERS analysis with machine learning is a potential diagnostic tool for LTBI screening.

Metal Oxide Nanostructures Enhanced Microfluidic Platform for Efficient and Sensitive Immunofluorescence Detection of Dengue Virus.

Rapid and sensitive detection of Dengue virus remains a critical challenge in global public health. This study presents the development and evaluation of a Zinc Oxide nanorod (ZnO NR)-surface-integrated microfluidic platform for the early detection of Dengue virus. Utilizing a seed-assisted hydrothermal synthesis method, high-purity ZnO NRs were synthesized, characterized by their hexagonal wurtzite structure and a high surface-to-volume ratio, offering abundant binding sites for bioconjugation. Further, a comparative analysis demonstrated that the ZnO NR substrate outperformed traditional bare glass substrates in functionalization efficiency with 4G2 monoclonal antibody (mAb). Subsequent optimization of the functionalization process identified 4% (3-Glycidyloxypropyl)trimethoxysilane (GPTMS) as the most effective surface modifier. The integration of this substrate within a herringbone-structured microfluidic platform resulted in a robust device for immunofluorescence detection of DENV-3. The limit of detection (LOD) for DENV-3 was observed to be as low as 3.1 × 10-4 ng/mL, highlighting the remarkable sensitivity of the ZnO NR-integrated microfluidic device. This study emphasizes the potential of ZnO NRs and the developed microfluidic platform for the early detection of DENV-3, with possible expansion to other biological targets, hence paving the way for enhanced public health responses and improved disease management strategies.

Highly cytocompatible and flexible three-dimensional graphene/polydimethylsiloxane composite for culture and electrochemical detection of L929 fibroblast cells.

Recently, three-dimensional graphene interconnected network has attracted great interest as a scaffold structure for tissue engineering due to its high biocompatibility, high electrical conductivity, high specific surface area and high porosity. However, free-standing three-dimensional graphene exhibits poor flexibility and stability due to ease of disintegration during processing. In this work, three-dimensional graphene is composited with polydimethylsiloxane to improve the structural flexibility and stability by a new simple two-step process comprising dip coating of polydimethylsiloxane on chemical vapor deposited graphene/Ni foam and wet etching of nickel foam. Structural characterizations confirmed an interconnected three-dimensional multi-layer graphene structure with thin polydimethylsiloxane scaffold. The composite was employed as a substrate for culture of L929 fibroblast cells and its cytocompatibility was evaluated by cell viability (Alamar blue assay), reactive oxygen species production and vinculin immunofluorescence imaging. The result revealed that cell viability on three-dimensional graphene/polydimethylsiloxane composite increased with increasing culture time and was slightly different from a polystyrene substrate (control). Moreover, cells cultured on three-dimensional graphene/polydimethylsiloxane composite generated less ROS than the control at culture times of 3-6 h. The results of immunofluorescence staining demonstrated that fibroblast cells expressed adhesion protein (vinculin) and adhered well on three-dimensional graphene/polydimethylsiloxane surface. Good cell adhesion could be attributed to suitable surface properties of three-dimensional graphene/polydimethylsiloxane with moderate contact angle and small negative zeta potential in culture solution. The results of electrochemical study by cyclic voltammetry showed that an oxidation current signal with no apparent peak was induced by fibroblast cells and the oxidation current at an oxidation potential of +0.9 V increased linearly with increasing cell number. Therefore, the three-dimensional graphene/polydimethylsiloxane composite exhibits high cytocompatibility and can potentially be used as a conductive substrate for cell-based electrochemical sensing.

Real-time multianalyte biosensors based on interference-free multichannel monolithic quartz crystal microbalance.

In this work, we design, fabricate and characterize a new interference-free multichannel monolithic quartz crystal microbalance (MQCM) platform for bio-sensing applications. Firstly, interference due to thickness-shear vibration mode coupling between channels in MQCM array is effectively suppressed by interposing a polydimethylsiloxane wall between adjacent QCM electrodes on a quartz substrate to form inverted-mesa-like structure. In addition, the electrical coupling due to the electrical impedance of solution is diminished by extending the flow path between them with an extended-design flow channel. The electrical testing results show that individual QCM signal is unaffected by those of adjacent channels under liquid loading, signifying the achievement of interference-free MQCM. The MQCM is applied for multi-analyte biosensing of IgG and HSA. The anti-IgG and anti-HSA are separately immobilized on two adjacent QCM electrodes, which are subsequently blocked with BSA to avoid unspecific binding. The MQCM biosensors are tested with single- and double-analyte solutions under continuous flow of buffer. The IgG and HSA QCM sensors only show frequency shift responses to their corresponding analytes and there are very small cross frequency shifts due to remnant unspecific binding. Moreover, MQCM sensors show approximately linear frequency shift response with analyte concentration. Therefore, the developed MQCM platform is promising for real-time interference-free label-free detection and quantification of multiple bio-analytes.

Increased oxidative DNA damage seen in renal biopsies adjacent stones in patients with nephrolithiasis.

Urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, is significantly higher in nephrolithiasis patients than in healthy individuals, indicating that these patients have higher degree of oxidative stress. In the present study, we investigated 8-OHdG expression in renal biopsies of patients with nephrolithiasis and in renal tubular cells (HK-2 cells) exposed to calcium oxalate monohydrate (COM). We performed immunohistochemical staining for 8-OHdG in renal biopsies adjacent stones obtained from 28 patients with nephrolithiasis. Controls were noncancerous renal tissues from nephrectomies of patients with renal cancer. 8-OHdG was overexpressed in the nucleus of renal tubular cells in patients with nephrolithiasis compared with controls. Only one nephrolithiasis biopsy was negative for 8-OHdG, whereas in 19 cases 8-OHdG was highly expressed. The level of expression of 8-OHdG among patients with calcium oxalate (mostly mixed with calcium phosphate) and uric acid stones was not significantly different. Increased leukocyte infiltration was observed in renal tissues from patients with nephrolithiasis. Exposure of HK-2 cells to COM caused increased intracellular reactive oxygen species and nuclear expression of 8-OHdG. To our knowledge, this is the first report of increased 8-OHdG expression in renal tubular cells of patients with nephrolithiasis. In vitro, COM crystals were capable of inducing oxidative damage of DNA in the proximal renal tubular cells.