Wide-field computational imaging of pathology slides using lens-free on-chip microscopy.

Optical examination of microscale features in pathology slides is one of the gold standards to diagnose disease. However, the use of conventional light microscopes is partially limited owing to their relatively high cost, bulkiness of lens-based optics, small field of view (FOV), and requirements for lateral scanning and three-dimensional (3D) focus adjustment. We illustrate the performance of a computational lens-free, holographic on-chip microscope that uses the transport-of-intensity equation, multi-height iterative phase retrieval, and rotational field transformations to perform wide-FOV imaging of pathology samples with comparable image quality to a traditional transmission lens-based microscope. The holographically reconstructed image can be digitally focused at any depth within the object FOV (after image capture) without the need for mechanical focus adjustment and is also digitally corrected for artifacts arising from uncontrolled tilting and height variations between the sample and sensor planes. Using this lens-free on-chip microscope, we successfully imaged invasive carcinoma cells within human breast sections, Papanicolaou smears revealing a high-grade squamous intraepithelial lesion, and sickle cell anemia blood smears over a FOV of 20.5 mm(2). The resulting wide-field lens-free images had sufficient image resolution and contrast for clinical evaluation, as demonstrated by a pathologist's blinded diagnosis of breast cancer tissue samples, achieving an overall accuracy of ~99%. By providing high-resolution images of large-area pathology samples with 3D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings.

Simultaneous detection of lactate and glucose by integrated printed circuit board based array sensing chip.

An integrated printed circuit board (PCB) based array sensing chip was developed to simultaneously detect lactate and glucose in mouse serum. The novelty of the chip relies on a concept demonstration of inexpensive high-throughput electronic biochip, a chip design for high signal to noise ratio and high sensitivity by construction of positively charged chitosan/redox polymer Polyvinylimidazole-Os (PVI-Os)/carbon nanotube (CNT) composite sensing platform, in which the positively charged chitosan/PVI-Os is mediator and electrostatically immobilizes the negatively charged enzyme, while CNTs function as an essential cross-linker to network PVI-Os and chitosan due to its negative charged nature. Additional electrodes on the chip with the same sensing layer but without enzymes were prepared to correct the interferences for high specificity. Low detection limits of 0.6 µM and 5 µM were achieved for lactate and glucose, respectively. This work could be extended to inexpensive array sensing chips with high sensitivity, good specificity and high reproducibility for various sensor applications.

Toner and paper-based fabrication techniques for microfluidic applications.

The interest in low-cost microfluidic platforms as well as emerging microfabrication techniques has increased considerably over the last years. Toner- and paper-based techniques have appeared as two of the most promising platforms for the production of disposable devices for on-chip applications. This review focuses on recent advances in the fabrication techniques and in the analytical/bioanalytical applications of toner and paper-based devices. The discussion is divided in two parts dealing with (i) toner and (ii) paper devices. Examples of miniaturized devices fabricated by using direct-printing or toner transfer masking in polyester-toner, glass, PDMS as well as conductive platforms as recordable compact disks and printed circuit board are presented. The construction and the use of paper-based devices for off-site diagnosis and bioassays are also described to cover this emerging platform for low-cost diagnostics.

Webcam camera as a detector for a simple lab-on-chip time based approach.

A modification of a webcam camera for use as a small and low cost detector was demonstrated with a simple lab-on-chip reactor. Real time continuous monitoring of the reaction zone could be done. Acid-base neutralization with phenolphthalein indicator was used as a model reaction. The fading of pink color of the indicator when the acidic solution diffused into the basic solution zone was recorded as the change of red, blue and green colors (%RBG.) The change was related to acid concentration. A low cost portable semi-automation analysis system was achieved.

High-throughput analysis of gangliosides in defined regions of fetal brain by fully automated chip-based nanoelectrospray ionization multi-stage mass spectrometry.

Gangliosides (GGs), a large group of sialylated glycosphingolipids, are considered biomarkers of human brain development, aging and certain diseases. Determination of individual GG components in complex mixtures extracted from a human brain represents a fundamental prerequisite for correlating their specificity with the specialized function of each brain area. In the context of modern glycomics, detailed investigation of GG expression and structure in human brain requires a continuous development and application of innovative methods able to improve the quality of data and speed of analysis. In this work, for the first time, a high-throughput mapping and sequencing of gangliosides in human fetal brain was performed by a novel mass spectrometry (MS)-based approach developed recently in our laboratory. Three GG mixtures extracted and purified from different regions of the same fetal brain in the 36th gestational week: frontal neocortex (NEO36), white matter of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) were subjected to comparative high-throughput screening and multi-stage fragmentation by fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) MS. Using this method, in only a few minutes of signal acquisitions, over 100 GG and asialo-GG species were detected and identified in the three mixtures. Obtained data revealed for the first time that differences in GG expression in human fetal brain are dependent on phylogenetic development rather than topographic factors. While a significant variation of GG distribution in NEO36 vs FL36 was observed, no significant differences in GG expression in white matter of frontal vs occipital lobe were detected. Additionally, the largest number of species was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. In the last stage of analysis, using MS(2)-MS(3) molecular ion fragmentation at variable amplitudes, a NEO36-associated GD1b isomer could clearly be discriminated. Present results indicate that the combination of fully automated chipESI with HCT MS(n) is able to provide ultra-fast, sensitive and reliable analyses of complex lipid-linked carbohydrates from which the pattern of their expression and structure in a certain type of bio-matrix can be determined.

Simple lab-on-chip approach with time-based detection.

A simple lab-on-chip approach with time-based detection is proposed. A platform is made from a piece of acrylic differently shaped channels for introducing sample and reagent(s) using flow manipulation. Time-based changes involving migration of the reaction zone are monitored. The changes can be visually monitored by using a stop-watch with naked eyes observation. Some applications for the determination of ascorbic acid, acetic acid and iron in real samples with different chemistries were demonstrated.

Nanostructured biosensing platform-shadow edge lithography for high-throughput nanofabrication.

One of the critical challenges in nanostructured biosensors is to manufacture an addressable array of nanopatterns at low cost. The addressable array (1) provides multiplexing for biomolecule detection and (2) enables direct detection of biomolecules without labeling and amplification. To fabricate such an array of nanostructures, current nanolithography methods are limited by the lack of either high throughput or high resolution. This paper presents a high-resolution and high-throughput nanolithography method using the compensated shadow effect in high-vacuum evaporation. The approach enables the fabrication of uniform nanogaps down to 20 nm in width across a 100 mm silicon wafer. The nanogap pattern is used as a template for the routine fabrication of zero-, one-, and two-dimensional nanostructures with a high yield. The method can facilitate the fabrication of nanostructured biosensors on a wafer scale at a low manufacturing cost.

A polymer lab-on-a-chip for magnetic immunoassay with on-chip sampling and detection capabilities.

This paper presents a new polymer lab-on-a-chip for magnetic bead-based immunoassay with fully on-chip sampling and detection capabilities, which provides a smart platform of magnetic immunoassay-based lab-on-a-chip for point-of-care testing (POCT) toward biochemical hazardous agent detection, food inspection or clinical diagnostics. In this new approach, the polymer lab-on-a-chip for magnetic bead-based immunoassay consists of a magnetic bead-based separator, an interdigitated array (IDA) micro electrode, and a microfluidic system, which are fully incorporated into a lab-on-a-chip on cyclic olefin copolymer (COC). Since the polymer lab-on-a-chip was realized using low cost, high throughput polymer microfabrication techniques such as micro injection molding and hot embossing method, a disposable polymer lab-on-a-chip for the magnetic bead-based immunoassay can be successfully realized in a disposable platform. With this newly developed polymer lab-on-a-chip, an enzyme-labelled electrochemical immunoassay (ECIA) was performed using magnetic beads as the mobile solid support, and the final enzyme product produced from the ECIA was measured using chronoamperometry. A sampling and detection of as low as 16.4 ng mL(-1) of mouse IgG has been successfully performed in 35 min for the entire procedure.

Lab-on-a-chip technologies for oral-based cancer screening and diagnostics: capabilities, issues, and prospects.

The design of a microfluidic lab-on-a-chip system for point-of-care cancer screening and diagnosis of oral squamous cell carcinoma (OSCC) is presented. The chip is based on determining a approximately 30-gene transcription profile in cancer cells isolated from oral fluid samples. Microfluidic cell sorting using magnetic beads functionalized with an antibody against cancer-specific cell-surface antigens (e.g., epithelial cell adhesion molecule [EpCAM]) is described. A comprehensive cancer diagnostics chip will integrate microfluidic components for cell lysis, nucleic acid extraction, and amplification and detection of a panel of mRNA isolated from a subpopulation of cancer cells contained in a clinical specimen.

Macromolecular crystallization with microfluidic free-interface diffusion.

Fluidigm Corp. released the Topaz 1.96 and 4.96 crystallization chips in the fall of 2004. Topaz 1.96 and 4.96 are the latest evolution of Fluidigm's microfluidics crystallization technologies that enable ultra-low-volume rapid screening for macromolecular crystallization. Topaz 1.96 and 4.96 are similar to each other but represent a major redesign of the Topaz system and have substantially improved ease of automation and ease of use, improved efficiency and even further reduced the amount of material needed. With the release of the new Topaz system, Fluidigm continues to set the standard in low-volume crystallization screening, which is having an increasing impact in the field of structural genomics and more generally in structural biology. It is likely that further optimization and increased utility of the Topaz crystallization system will emerge. It is also probable that further innovation and the emergence of competing technologies will be seen.