Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using molecular inversion probe sequencing and breakpoint-spanning polymerase chain reaction analysis we reliably detected alterations with an allele frequency below 1%. We identified 660 genomic alterations in 285 diagnostic samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.Development and application of statistical models for medical scientific researc

Ion-Transfer Voltammetric Behavior of Propranolol at Nanoscale Liquid-Liquid Interface Arrays

In this work, the ion-transfer voltammetric detection of the protonated β-blocker propranolol was explored at arrays of nanoscale interfaces between two immiscible electrolyte solutions (ITIES). Silicon nitride nanoporous membranes with 400 pores in a hexagonal arrangement, with either 50 or 17 nm radius pores, were used to form regular arrays of nanoITIES. It was found that the aqueous-to-organic ion-transfer current continuously increased steadily rather than reaching a limiting current plateau after the ion-transfer wave; the slope of this limiting current region was concentration dependent and associated with the high ion flux at the nanointerfaces. Electrochemical data were examined in terms of an independent nanointerface approach and an equivalent microdisc approach, supported by finite element simulation. In comparison to the larger interface configuration (50 nm radius), the array of 17 nm radius nanoITIES exhibited a 6.5-times higher current density for propranolol detection due to the enhanced ion flux arising from the convergent diffusion to smaller electrochemical interfaces. Both nanoITIES arrays achieved the equivalent limits of detection, 0.8 μM, using cyclic voltammetry. Additionally, the effect of scan rate on the charging and faradaic currents at these nanoITIES arrays, as well as their stability over time, was investigated. The results demonstrate that arrays of nanoscale liquid–liquid interfaces can be applied to study electrochemical drug transfer, and provide the basis for the development of miniaturized and integrated detection platforms for drug analysis

Lab-on-a-chip technology for clinical diagnostics: The fertility chip

In the 1990s the term micro total analysis systems (μTAS) was introduced to describe a complete microsystem which integrates sample handling, analysis and detection into a single device, also called Lab-on-a-Chip (LOC) device (1). The LOC concept defines the scaling down of a single or multiple lab processes into a chip format with dimensions as small as a stamp. Scaling down offers many advantages, such as less sample, reagent and waste volumes, faster analysis, integration of many analytical processes within one device, lower cost, to name a few, but first of all an easy handling. These advantages meet the actual demands of clinical laboratories, which are dealing with an increasing workload and decreased funding. Our group showed previously these advantages of the LOC technology for blood electrolyte determinations in clinical diagnostics (2). Furthermore, microfluidic dimensions (10 - 100 μm) equal the size of cells, making these devices very suitable for the analysis of many different biochemical processes even on a single-cell level. Hence, there are many reasons why microtechnology is advantageous compared to existing conventional analysis methods, especially in the case of cellular based assays, to understand how cells react in a certain environment, to a certain drug or in contact with other cell types. Different cell manipulation methods (e.g. sorting, detachment, staining, fixing, lysis) can be integrated on one chip, less sample is needed ideally when only a few cells are available (e.g., primary cells) and the dimensions favour single-cell analysis. Furthermore, optical detection techniques can be automated and in some cases be replaced by electrical on-chip detection methods. Moreover, development of cell arrays, which are analogous to DNA or protein arrays, offer the possibility for high-throughput screening. Recent technological developments enable detailed cellular studies, defining a new concept: Lab-in-a-Cell. In this concept the cell is used as a laboratory to perform complex biological operations. Micro- and even nanotechnological tools are employed to access and analyse this laboratory and interface it with the outside world. In the present manuscript we will summarize our recent efforts to demonstrate the advantages of LOC technology to study cells for clinical diagnostics by working on a fertility chip as an example

Increasing the Sensitivity of Electrochemical DNA Detection by a Micropillar-Structured Biosensing Surface

The available active surface area and the density of probes immobilized on this surface are responsible for achieving high specificity and sensitivity in electrochemical biosensors that detect biologically relevant molecules, including DNA. Here, we report the design of gold-coated, silicon micropillar-structured electrodes functionalized with modified poly-l-lysine (PLL) as an adhesion layer to concomitantly assess the increase in sensitivity with the increase of the electrochemical area and control over the probe density. By systematically reducing the center-to-center distance between the pillars (pitch), denser micropillar arrays were formed at the electrode, resulting in a larger sensing area. Azido-modified peptide nucleic acid (PNA) probes were click-reacted onto the electrode interface, exploiting PLL with appended oligo(ethylene glycol) (OEG) and dibenzocyclooctyne (DBCO) moieties (PLL-OEG-DBCO) for antifouling and probe binding properties, respectively. The selective electrochemical sandwich assay formation, composed of consecutive hybridization steps of the target complementary DNA (cDNA) and reporter DNA modified with the electroactive ferrocene functionality (rDNA-Fc), was monitored by quartz crystal microbalance. The DNA detection performance of micropillared electrodes with different pitches was evaluated by quantifying the cyclic voltammetric response of the surface-confined rDNA-Fc. By decrease of the pitch of the pillar array, the area of the electrode was enhanced by up to a factor 10.6. A comparison of the electrochemical data with the geometrical area of the pillared electrodes confirmed the validity of the increased sensitivity of the DNA detection by the design of the micropillar array

Microbubbles for medical applications

Ultrasound contrast agent (UCA) suspensions contain encapsulated microbubbles with radii ranging from 1 to 10 micrometers. The bubbles oscillate to the driving ultrasound pulse generating harmonics of the driving ultrasound frequency. This feature allows for the discrimination of non-linear bubble echoes from linear tissue echoes facilitating the visualization and quantification of blood perfusion in organs. Targeting the microbubbles to specific receptors in the body has led to molecular imaging application with ultrasound and targeted drug delivery with drug-loaded microbubbles. Traditional UCA production methods offer high yield but poor control over the microbubble size and uniformity. Medical ultrasound transducers typically operate at a single frequency, therefore only a small selection of bubbles resonates to the driving ultrasound pulse. Here we discuss recent lab-on-a-chip based production and sorting methods that have been shown to produce highly monodisperse bubbles, thereby improving the sensitivity of contrast-enhanced ultrasound imaging and molecular imaging with microbubbles. Moreover, monodisperse UCA show great potential for targeted drug delivery by the well-controlled bubble response

Magnetic particle actuation in stationary microfluidics for integrated lab-on-chip biosensors

The aging population and increases in chronic diseases put high pressure on the healthcare system, which drives a need for easy-to-use and cost-effective medical technologies. In-vitro diagnostics (IVD) plays a large role in delivering healthcare and, within the IVD market, decentralized diagnostic testing, i.e. point-of-care testing (POCT), is a growing segment. POCT devices should be compact and fully integrated for maximum ease of use. A new class of POCT technologies is appearing based on actuated magnetic particles. The use of magnetic particles has important advantages: they have a large surface-to-volume ratio, are conveniently biofunctionalized, provide a large optical contrast, and can be manipulated by magnetic fields. In this chapter, we review the use of magnetic particles actuated by magnetic fields to realize integrated lab-on-chip diagnostic devices wherein several assay process steps are combined, e.g. to mix fluids, capture analytes, concentrate analytes, transfer analytes, label analytes, and perform stringency steps. We focus on realizations within the concept of stationary microfluidics and we discuss efforts to integrate different magnetically actuated assay steps, with the vision that it will become possible to realize biosensing systems in which all assay process steps are controlled and optimized by magnetic forces

Inverse-woodpile photonic band gap crystals with a cubic diamond-like structure made from single-crystalline silicon

Three dimensional photonic band gap crystals with a cubic diamond-like symmetry are fabricated. These so-called inverse-woodpile nanostructures consist of two perpendicular sets of pores in single-crystal silicon wafers and are made by means of complementary metal oxide-semiconductor (CMOS)-compatible methods. Both sets of pores have high aspect ratios and are made by deep reactive-ion etching. The mask for the first set of pores is defined in chromium by means of deep UV scan-and-step technology. The mask for the second set of pores is patterned using an ion beam and carefully placed at an angle of 90°with an alignment precision of better than 30 nm. Crystals are made with pore radii between 135-186 nm with lattice parameters a = 686 and c = 488 nm such that a/c = √2; hence the structure is cubic. The crystals are characterized using scanning electron microscopy and X-ray diffraction. By milling away slices of crystal, the pores are analyzed in detail in both directions regarding depth, radius, tapering, shape, and alignment. Using optical reflectivity it is demonstrated that the crystals have broad reflectivity peaks in the near-infrared frequency range, which includes the telecommunication range. The strong reflectivity confirms the high quality of the photonic crystals. Furthermore the width of the reflectivity peaks agrees well with gaps in calculated photonic band structures