Microfluidic-based diagnostics for cervical cancer cells

The use of biomarkers has facilitated the detection of specific tumor cells. However, the technology to apply these markers in a clinical setting has not kept pace with their increasing availability. In this project, we use an antibody-based microfluidics platform to recognize and capture cervical cancer cells. Because HPV-16 infection of cervical cells and up-regulation of alpha6-integrin cell surface receptors are correlated, we utilized alpha6-integrin as a capture antibody bound to the channel surface. Normal human glandular epithelial cells (HGEC), human cervical stromal cells (HCSC) and cervical cancer cells (HCCC) were suspended in PBS and flowed through the system. Greater than 30% of the cancer cells were captured while the capture of the normal cell types was less than 5%. The technique is sensitive and accurate. It is potentially useful in the detection of cervical cancer at all stages, as well as other of cancers with similar characteristics of cell surface antigen expression.     

Novel nucleoside triphosphate analogs for the enzymatic labeling of nucleic acids

We have evaluated several novel nucleotide analogs suitable for enzymatic labeling of nucleic acid targets for a variety of array-based assays. Two new reagents in particular, a C4-labeled 1-(2',3'-dideoxy-beta-D-ribofuranosyl) imidazole-4-carboxamide 5'-triphosphate 5 and an N1-labeled 5-(beta-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5'-triphosphate 3, were found to be excellent substrates for labeling with terminal deoxynucleotidyl transferase and T7 RNA polymerase, respectively.     

Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.     

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.Download video file.^{(62M, mov)}     

Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.     

pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.

We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.     

Usefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on-chip enzymatic digestion.

The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.     

miRNAs

The evolution of complex animals such as insects and mammals is achieved with surprisingly few additions in protein coding genes. MicroRNAs (miRNAs), a class of non-coding RNAs, have emerged as important regulators of organogenesis in insects, fish and mammals. The microRNA repertoire of animals has expanded significantly during evolution especially in vertebrates, insects and nematodes, accompanying the appearance of complex body plans. MicroRNAs therefore have gained enormous interest in recent years. They are now regarded as key modulators of gene expression in many tissues during embryogenesis, in adult organisms and in disease processes. Therefore, these small RNA molecules have entered the center stage of molecular biology and are promising candidates not only for the regulation of key biological processes such as proliferation and apoptosis, but also for therapy of human diseases.     

Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.     

Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics

Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood. We present a microfluidic-based, sample-preparation system that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.     

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.     

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson''s disease (PD), which involves the degeneration of dopaminergic (DA) neurons ¹. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds ^2,3.Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans'' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") ⁴. Reversing the field''s polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response ⁵. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae ⁴. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype ⁶.We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae ⁷. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel ⁸.Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.     

Triple isotopic labeling and kinetic isotope effects: exposing H-transfer steps in enzymatic systems

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.     

Production of frameshift mutations in Salmonella by phenanthridinium derivatives: enzymatic activation and photoaffinity labeling

The effect of metabolic activation on the mutagenic potential of some phenanthridinium compounds was examined in Salmonella typhimurium strains TA1538 and TA1978 . All of the compounds tested were mutagenic in TA1538, a DNA excision-repair-deficient strain, when metabolizing enzymes were included in the assay. Reversions were not detected when these compounds were examined under the same conditions in TA1978 , the isogenic strain of TA1538 proficient in DNA repair. The mutagenic activity of an azido analog of propidium iodide was also examined using photoactivation and enzymatic activation, and with both conditions, reversions were observed in TA1538 but not in TA1978 . Furthermore, the ranking of mutagenic activity of propidium azide relative to ethidium azide analogs was comparable for both types of activation. The evidence from several studies suggests that the structural requirements for mutagenic activity for this series of phenanthridinium compounds appear to be the same whether mutagenesis is induced via photoactivation or metabolic activation. The interaction with DNA resulting in covalent alteration of the DNA is implicated as the mutagenic mechanism whether the active species is generated by metabolic- or photo-activation.