Light emitting diode, photodiode-based fluorescence detection system for DNA analysis with microchip electrophoresis.

Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity.

An optical relay approach to very low cost hybrid polymer-complementary metal-oxide semiconductor electrophoresis instrumentation.

Electrophoresis is an integral part of many molecular diagnostics protocols and an inexpensive implementation would greatly facilitate point-of-care (POC) applications. However, the high instrumentation cost presents a substantial barrier, much of it associated with fluorescence detection. The cost of such systems could be substantially reduced by placing the fluidic channel and photodiode directly above the detector in order to collect a larger portion of the fluorescent light.

Millimeter scale separation of DNA with a replaceable polymer matrix.

Electrophoresis is a powerful method that has seen a wide range of applications, often in automated genetic diagnostic instruments that require the use of a replaceable sieving matrix. The power and simplicity of electrophoresis as an analysis technique would be ideal for highly integrated and low-cost analysis systems if the method could be implemented in microfluidics on the scale of several mm. We demonstrate the electrophoretic analysis of DNA with separation lengths as small as 2 mm and with a resolution adequate for the analysis of PCR products, i.

Towards robust cellular image classification: theoretical foundations for wide-angle scattering pattern analysis.

Clinical analysis of light scattering from cellular organelle distributions can help identify disease and predict a patient's response to treatment. This work presents a theoretical basis for the identification of important intracellular distributions from scattering patterns even in the presence of optical and structural variability, and examines how the geometry of an organelle distribution affects key properties of wide-angle (two-dimensional) scattering patterns. Specifically, this work demonstrates how organelle arrangement relates to the size and shape of intensity peaks within simulated scattering images, and how this relationship can affect cell identification when using standard image classification methods.

Development of a microfluidic chip-based plasmid miniprep.

Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16h, including the time needed for bacterial cell culture.

Strategies for enhancing the speed and integration of microchip genetic amplification.

In this work, we explore the use of methods that allow a significant acceleration of genetic analysis within microchips fabricated from low thermal conductivity materials such as glass or polymers. Although these materials are highly suitable for integrating a number of genetic analysis techniques onto lab-on-a-chip devices, their low thermal conductivity limits the rate at which heat can be transferred and hence lowers the speed of thermal cycling. However, short thermal cycling times are the key to bringing PCR to clinical point-of-care applications.

An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique for probing the genetic content of individual cells at the chromosomal scale. Conventional FISH techniques provide a sensitive diagnostic tool for the detection of chromosomal alterations on a cell-by-cell basis; however, the cost-per-test in terms of reagent and highly qualified labour has prevented its wide-spread utilization in clinical settings. Here, we address the inefficient use of labour with the first integrated and automated on-chip FISH implementation, one that requires only minutes of setup time from the technician.

Rapid simulation of wide-angle scattering from mitochondria in single cells.

It has been shown that the mitochondria are the dominant source of large-angle light scattering from human cells. In the limit of small mitochondria, we show that the large-angle (isotropic) light scattering of mitochondria may be analyzed and simulated with an adaptation of classical X-ray diffraction theory. In addition, we show that this approach may be extended to the case of anisotropic scatter.

Electrically controlled microvalves to integrate microchip polymerase chain reaction and capillary electrophoresis.

Microvalves are key in realizing portable miniaturized diagnostic platforms. We present a scalable microvalve that integrates well with standard lab on a chip (LOC) implementations, yet which requires essentially no external infrastructure for its operation. This electrically controlled, phase-change microvalve is used to integrate genetic amplification and analysis via capillary electrophoresis--the basis of many diagnostics.

A microfluidic study of mechanisms in the electrophoresis of supercoiled DNA.

In this work, microfluidic chips were used to study the electrophoresis of supercoiled DNA (SC DNA) in agarose. This system allowed us to study the electrophoretic and trapping behaviours of SC DNA of various lengths, at various fields and separation distances. Near a critical electric field the DNA is trapped such that the concentration falls exponentially with distance.

Dynamic temperature measurement in microfluidic devices using thermochromic liquid crystals.

Thermochromic liquid crystals (TLCs) are used to explore the temperature transients during thermal cycling for microchip-based polymerase chain reaction (PCR). By analyzing the reflected spectra of the TLCs over time, temperature vs. time trajectories were extracted and overshoots/undershoots were estimated.

An inexpensive and portable microchip-based platform for integrated RT-PCR and capillary electrophoresis.

We present an inexpensive, portable and integrated microfluidic instrument that is optimized to perform genetic amplification and analysis on a single sample. Biochemical reactions and analytical separations for genetic analysis are performed within tri-layered glass-PDMS microchips. The microchip itself consists of integrated pneumatically-actuated valves and pumps for fluid handling, a thin-film resistive element that acts simultaneously as a heater and a temperature sensor, and channels for capillary electrophoresis (CE).

Integrated wavelength-selective optical waveguides for microfluidic-based laser-induced fluorescence detection.

We demonstrate the fabrication and characterization of a novel, inexpensive microchip capable of laser induced fluorescence (LIF) detection using integrated waveguides with built-in optical filters. Integrated wavelength-selective optical waveguides are fabricated by doping poly(dimethysiloxane) (PDMS) with dye molecules. Liquid-core waveguides are created within dye-doped PDMS microfluidic chips by filling channels with high refractive index liquids.

Rapid fabrication of a microfluidic device with integrated optical waveguides for DNA fragment analysis.

The fabrication and performance of a microfluidic device with integrated liquid-core optical waveguides for laser induced fluorescence DNA fragment analysis is presented. The device was fabricated through poly(dimethylsiloxane) (PDMS) soft lithography and waveguides are formed in dedicated channels through the addition of a liquid PDMS pre-polymer of higher refractive index. Once a master has been fabricated, microfluidic chips can be produced in less than 3 h without the requirement for a cleanroom, yet this method provides an optical system that has higher performance than a conventional confocal optical assembly.

Microfluidic platform for single nucleotide polymorphism genotyping of the thiopurine S-methyltransferase gene to evaluate risk for adverse drug events.

Prospective clinical pharmacogenetic testing of the thiopurine S-methyltransferase gene remains to be realized despite the large body of evidence demonstrating clinical benefit for the patient and cost effectiveness for health care systems. We describe an entirely microchip-based method to genotype for common single nucleotide polymorphisms in the thiopurine S-methyltransferase gene that lead to serious adverse drug reactions for patients undergoing thiopurine therapy. Restriction fragment length polymorphism and allele-specific polymerase chain reaction have been adapted to a microfluidic chip-based polymerase chain reaction and capillary electrophoresis platform to genotype the common *2, *3A, and *3C functional alleles.

Microfluidic chips for detecting the t(4;14) translocation and monitoring disease during treatment using reverse transcriptase-polymerase chain reaction analysis of IgH-MMSET hybrid transcripts.

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis.

Rapid prototyping of microfluidic devices with a wax printer.

We demonstrate a rapid and inexpensive approach for the fabrication of high resolution poly(dimethylsiloxane) (PDMS)-based microfluidic devices. The complete process of fabrication could be performed in several hours (or less) without any specialized equipment other than a consumer-grade wax printer. The channels produced by this method are of high enough quality that we are able to demonstrate the sizing and separation of DNA fragments using capillary electrophoresis (CE) with no apparent loss of resolution over that found with glass chips fabricated by conventional photolithographic methods.

A method for cytometric image parameterization.

New advances in wide-angle cytometry have allowed researchers to obtain micro- and nano-structural information from biological cells. While the complex two-dimensional scattering patterns generated by these devices contain vital information about the structure of a cell, no computational analysis methods have been developed to rapidly extract this information. In this work we demonstrate a multi-agent computational pipeline that is able to extract features from a two-dimensional laser scattering image, cluster these features into spatially distinct regions, and extract a set of parameters relating to the structure of intensity regions within the image.

Automated screening using microfluidic chip-based PCR and product detection to assess risk of BK virus-associated nephropathy in renal transplant recipients.

The cost-effective detection of viral particles in bodily fluids could enable more effective responses to viral outbreaks, whether isolated clinical cases, or influenza epidemics. In renal transplant recipients, complications arising from high levels of BK virus can lead to graft dysfunction, graft loss, and/or reduced patient survival. We describe a microfluidic system for the sensitive analysis of BK virus (viral load) in unprocessed urine samples that are applied directly onto the chip, thus avoiding labor-intensive processing and sources of inter-assay variability.

A miniaturized wide-angle 2D cytometer.

Background: We present an optical waveguide based cytometer that is capable of simultaneously collecting the light scattered by cells over a wide range of solid angles. Such comprehensive scattering data are a prerequisite for the microstructural characterization of cells.

Methods: We use latex beads as cell mimics, and demonstrate the ability of this new cytometer to collect back-scattered light in two dimensions (2D).

Rapid on-chip postcolumn labeling and high-resolution separations of DNA.

When performing genetic analysis on microfluidic systems, labeling the sample DNA for detection is a critical preparation step. Labeling procedures often involve fluorescently tagged primers and PCRs, which lengthen experimental run times and introduce higher levels of complexity, increasing the overall cost per analysis. Alternatively, on-chip labeling techniques based on intercalating dyes permit rapid labeling of DNA fragments.

An adaptable microvalving system for on-chip polymerase chain reactions.

On-chip genetic analysis systems are beginning to provide a viable alternative to conventional gene profiling and amplification devices, through minimal reagent use, high detection resolution, and the potential for high-throughput parallel testing of the genetic material, even from single cells. Despite the advantages, there are many difficulties inherent in creating an integrated microfluidic diagnostic platform. One major challenge is the accurate control and manipulation of fluid, and particularly the immobilization of reaction mixtures during heating phases of polymerase chain reactions (PCR).

Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.

Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma.

Analysis of mitochondrial DNA in microfluidic systems.

Abnormalities in mitochondrial function play a major role in many human diseases. It is often of critical importance to ascertain what proportion of the mitochondria within a cell, or cells, bear a given mutation (the mitochondrial "demographics"). In this work, a rapid, novel, on-chip procedure was used, in which a restriction enzyme was employed to excise a mitochondrial DNA (mtDNA) sequence from plasmid DNA that acted as a prototypical mitochondrial genome.

A rejuvenation method for poly(N,N-dimethylacrylamide)-coated glass microfluidic chips.

As microfluidic chips come to integrate the higher levels of functionality required for the implementation of advanced bioanalytical protocols, a crucial factor is that of cost. Although glass chips provide advantages in multilayer integrations, their cost is far higher than that of polymer chips. However, a simple and effective rejuvenation protocol for glass microchips may enable higher levels of integration and functionality on glass microchips.