Innovative PNA-LB mediated allele-specific LAMP for KRAS mutation profiling on a compact lab-on-a-disc device.

Tailored healthcare, an approach focused on individual patients, requires integrating emerging interdisciplinary technologies to develop accurate and user-friendly diagnostic tools. KRAS mutations, prevalent in various common cancers, are crucial determinants in selecting patients for novel KRAS inhibitor therapies. This study presents a novel state-of-the-art Lab-on-a-Disc system utilizing peptide nucleic acids-loop backward (PNA-LB) mediated allele-specific loop-mediated isothermal amplification (LAMP) for detecting the frequent G12D KRAS mutation, signifying its superiority over alternative mutation detection approaches. The designed Lab-on-a-Disc system demonstrated exceptional preclinical and technical precision, accuracy, and versatility. By applying varying cutoff values to PNA- LB LAMP reactions, the assay's sensitivity and specificity were increased by 80 % and 90 %, respectively. The device's key advantages include a robust microfluidic Lab-on-a-Disc design, precise rotary control, and a cutting-edge induction heating module. These features enable multiplexing of LAMP reactions with high reproducibility and repeatability, with CV% values less than 3.5 % and 5.5 %, respectively. The device offers several methods for accurate endpoint result detection, including naked-eye observation, RGB image analysis using Python code, and time of fluorescence (Tf) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence Tf values and pH-sensitive dyes, demonstrated comparable performance to the best standard methods. Overall, this study represents a significant step towards tailoring treatment strategies for cancer patients through precise and efficient mutation detection technologies.

An experimental study of centrifugal microfluidic platforms for magnetic-inertial separation of circulating tumor cells using contraction-expansion and zigzag arrays.

Cancer diagnosis has recently been at the forefront of recent medical research, with ongoing efforts to develop devices and technologies for detecting cancer in patients. One promising approach for cancer diagnosis is the detection of Circulating Tumor Cells (CTCs) in blood samples. Separating these rare cells from the diverse background of blood cells and analyzing them can provide valuable insights into the disease's stage and lethality. Here we present the design and fabrication of a centrifugal microfluidic platform on a polymeric disk that utilizes centrifugal forces for cell isolation. The separation units exploit both active and passive methods. In other words, in addition to introducing novel geometry for channels, an external magnetic field is also employed to separate the target cells from the background cells. In order for the external field to function, the CTCs must first be labeled with antibody-conjugated nanoparticles; the separation process should be then performed. Before the experimental tests, a numerical study was done to determine the optimum parameters; the angular velocity and magnetization investigations showed that 2000 rpm and 868,000 (kA/m) are the optimum conditions for the designed device to reach the efficiency of 100% for both White Blood Cells (WBCs) and CTCs. These results indicate that the passive region of the channels primarily contributes to the focusing of the target cells, and showed that the focusing effect is more pronounced in the expansion-contraction geometry compared to the zigzag geometry. Additionally, the results proved that curved channel geometries performed better than straight ones in terms of separation efficiency. However, if the separation relies solely on channel geometry, the majority of cells would be directed towards the non-target chamber, leading to suboptimal results. This is due to the direction of the forces acting on the cells. However, including an external magnetic field improves the direction of the net force and enhances the separation efficiency. Finally, the numerical and experimental results of the study were compared, and the curved expansion-contraction channel is introduced as the best geometry having 100% and ∼92% CTC separation efficiency, respectively.

Numerical study of a double-stair-shaped dielectrophoresis channel for continuous on-chip cell separation and lysis using finite element method.

Prognostication of numerous chronic diseases are in need of identifying circulating tumor cells (CTCs), afterwards, separating and reviving contaminated samples are required. Conventional methods of separating blood cells, namely cytometry or magnetically activated cell sorting, in many cases lose their functionality, or efficiency under different conditions. Hence microfluidic methods of separation have been implemented. Herein, an innovative integrated double stair-shaped microchannel is designed and optimized, capable of 'separation', and 'chemical lysis' simultaneously in which the lysis reagent concentration can be controlled to tune the lysis intensity. The method of insulator-based dielectrophoresis (iDEP), which is the main physics in this device, is utilized yielding maximum separation. Pivotal features of the applied voltage, the voltage difference, the angles and the number of stairs, and the width of the throat in the microchannel have been numerically explored in order to optimize the channel in terms of separation and the lysis buffer concentration. The overall state of optimum case for the voltage difference (ΔV) of 10 owns the following features: the number of stairs is 2, the angle of stairs is 110°, the width of throat is 140 µm, and the inlet voltages are 30 V and 40 V. Also, the overall state of optimum cases for delta possess the following features: the number of stairs is 2, the angle of stairs is 110°, the width of throat is 140 µm, and the inlet voltages are 30 V and 35 V.

Continuous Submicron Particle Separation Via Vortex-Enhanced Ionic Concentration Polarization: A Numerical Investigation.

Separation and isolation of suspended submicron particles is fundamental to a wide range of applications, including desalination, chemical processing, and medical diagnostics. Ion concentration polarization (ICP), an electrokinetic phenomenon in micro-nano interfaces, has gained attention due to its unique ability to manipulate molecules or particles in suspension and solution. Less well understood, though, is the ability of this phenomenon to generate circulatory fluid flow, and how this enables and enhances continuous particle capture. Here, we perform a comprehensive study of a low-voltage ICP, demonstrating a new electrokinetic method for extracting submicron particles via flow-enhanced particle redirection. To do so, a 2D-FEM model solves the Poisson-Nernst-Planck equation coupled with the Navier-Stokes and continuity equations. Four distinct operational modes (Allowed, Blocked, Captured, and Dodged) were recognized as a function of the particle's charges and sizes, resulting in the capture or release from ICP-induced vortices, with the critical particle dimensions determined by appropriately tuning inlet flow rates (200-800 [µm/s]) and applied voltages (0-2.5 [V]). It is found that vortices are generated above a non-dimensional ICP-induced velocity of U*=1, which represents an equilibrium between ICP velocity and lateral flow velocity. It was also found that in the case of multi-target separation, the surface charge of the particle, rather than a particle's size, is the primary determinant of particle trajectory. These findings contribute to a better understanding of ICP-based particle separation and isolation, as well as laying the foundations for the rational design and optimization of ICP-based sorting systems.