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The study explored the chronic toxicity of triclosan to green microalga Chlorococcum sp. under multiple interactions among multiple environmental conditions. This is the first study on chronic algal toxicity to combine synchrotron-based Fourier transform infrared spectromicroscopy, factorial analysis, principal component analysis, and stepwise-cluster analysis. Such a combination helps to reveal the toxic mechanism at the molecular level and explore the inner correlationship among multiple environmental conditions. In the 120-h test, nitrogen content became the most significant factor of the physiochemical properties. Some insignificant factors in the 48-h test became significant in the 120-h test. Temperature * nitrogen content, temperature * phosphorus content, and pH * phosphorus content were the most significant two-order interactions. Temperature * pH * NaCl concentration and temperature * NaCl concentration * phosphorus content were the most significant three-order interactions. More high-order interactions became significant in the 120-h test, indicating the complexity and impacts of all the factors may increase when time was extended. The chronic toxicity of triclosan presented more distinguishable variations among treatments based on biochemical alterations. These results demonstrate that the sensitivity and fragility of algae to triclosan can be amplified with time extension. Long-term exposure can be applied to better evaluate and predict the environmental toxicity behavior of triclosan. It can also help with environmental evaluation and risk management of real-world triclosan toxicity.

We present a microfluidic device for specifically capturing cancer cells and isolating their genomic DNA (gDNA) for specific amplification and sequence analysis. To capture cancer cells within the device, nucleic acid aptamers that specifically bind to cancer cells were immobilized within a channel containing micropillars designed to increase capture efficiency. The captured cells were lysed in situ, and their gDNA was isolated by physical entanglement within a second smaller-dimensioned micropillar array. This type of isolation allows the gDNA to be retained and purified within the channel and enables amplification and analysis to be performed on the gDNA without the loss of the original template. We developed a technique for selectively amplifying genes from whole gDNA using multiple displacement amplification. The amplified gene samples were sequenced, and the resulting sequence information was compared against the known wild-type gene to identify any mutations. We have tested cervical and ovarian cancer cells for mutations in the TP53 gene using this technology. This approach offers a way to monitor multiple genetic mutations in the same small population of cells, which is beneficial given the wide diversity in cancer cells, and therefore it requires very few cells to be extracted from a patient sample.

Target DNA fragments at 10 fM concentration (approximately 6 × 10(5) molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences.

Graphene's suite of useful properties makes it of interest for use in biosensors. However, graphene interacts strongly with hydrophobic components of biomolecules, potentially altering their conformation and disrupting their biological activity. We have immobilized the protein Concanavalin A onto a self-assembled monolayer of multivalent tripodal molecules on single-layer graphene. We used a quartz crystal microbalance (QCM) to show that tripod-bound Concanavalin A retains its affinity for polysaccharides containing α-D-glucopyrannosyl groups as well as for the α-D-mannopyranosyl groups located on the cell wall of Bacillus subtilis. QCM measurements on unfunctionalized graphene indicate that adsorption of Concanavalin A onto graphene is accompanied by near-complete loss of these functions, suggesting that interactions with the graphene surface induce deleterious structural changes to the protein. Given that Concanavalin A's tertiary structure is thought to be relatively robust, these results suggest that other proteins might also be denatured upon adsorption onto graphene, such that the graphene-biomolecule interface must be considered carefully. Multivalent tripodal binding groups address this challenge by anchoring proteins without loss of function and without disrupting graphene's desirable electronic structure.

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin—a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 108 with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays estimated to contain more than 250 000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are fixed in their extended configuration by adsorption onto the substrate so analysis can be performed with high spatial resolution and signal averaging. We further prove this technique allows imaging of DNA molecules with different methylation states.

Epigenetic states are governed by DNA methylation and a host of modifications to histones bound with DNA. These states are essential for proper developmentally regulated gene expression and are perturbed in many diseases. There is great interest in identifying epigenetic mark placement genome-wide and understanding how these marks vary among cell types, with changes in environment or according to health and disease status. Current epigenomic analyses employ bisulfite sequencing and chromatin immunoprecipitation, but query only one type of epigenetic mark at a time, DNA methylation or histone modifications, and often require substantial input material. To overcome these limitations, we established a method using nanofluidics and multi-color fluorescence microscopy to detect DNA and histones in individual chromatin fragments at about 10 Mbp/min. We demonstrated its utility for epigenetic analysis by identifying DNA methylation on individual molecules. This technique will provide the unprecedented opportunity for genome-wide, simultaneous analysis of multiple epigenetic states on single molecules using femtogram quantities of material.

The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores. To have meaningful interpretations of results from the study of labeled enzymes, it is then necessary to reduce the variability in physical, chemical, and biological characteristics of the labeled products. In this paper, a solid phase labeling protocol is described as an advantageous alternative to free solution labeling of cellulose-binding proteins and is applied to tag cellulases with three different fluorophores. The products from the labeling reactions were purified to remove the unreacted dye and separate labeled and unlabeled enzymes. Characterization of the catalytic and spectroscopic properties of the isolated labeled species confirmed that highly homogeneous populations of labeled cellulases can be achieved. The protocol for the separation of labeled products is applicable to any mixture of labeled proteins, making this an attractive methodology for the production of labeled proteins suitable for single molecule fluorescence spectroscopy.

We have demonstrated a new electrospray ionization (ESI) device incorporating a tip made from a shaped thin film, bonded to a microfluidic channel, and interfaced to a time-of-flight mass spectrometer (TOFMS). A triangular-shaped thin polymer tip was formed by lithography and etching. A microfluidic channel, 20 microm wide and 10 microm deep, was embossed in a cyclo olefin substrate using a silicon master. The triangular tip was aligned with the channel and bonded between the channel plate and a flat plate to create a microfluidic channel with a wicking tip protruding from the end. This structure aided the formation of a stable Taylor cone at the apex of the tip, forming an electrospray ionization source. This source was tested by spraying several solutions for mass spectrometric analysis. Because the components are all made by lithographic approaches with high geometrical fidelity, an integrated array system with multiple channels can be formed with the same method and ease as a single channel. We tested a multichannel system in a multiplexed manner and showed reliable operation with no significant cross contamination between closely spaced channels.

A novel technique that can rapidly separate long-strand polymers according to length is presented. The separation mechanism is mediated by a confinement-induced entropic force at the abrupt interface between regions of vastly different configuration entropy. To demonstrate this technique, DNA molecules were partially inserted into a dense array of nanopillars (an entropically unfavorable region) using a pulsed electric field and allowed to relax to their natural state by removal of the field. Molecules of dissimilar lengths (T2 and T7 coliphage DNA) were inserted into this region in such a way that shorter molecules were fully inserted in this region, while longer molecules remained partially across the interface. The longer T2 molecules were observed to recoil entirely out of the pillar array, leaving the shorter T7 molecules inserted, and effecting separation of the two species in a single step. To show how this method can be used for separation of unknown samples, the inserting electric field was pulsed for progressively longer times, allowing passage of progressively longer molecules and producing the equivalent of a conventional electropherogram. The effects limiting resolution in this device are discussed, and the expected separating power of a multistage device is reported. The extracted resolution and running separation time compare favorably with current conventional separation techniques.

A polymeric microfluidic chip made of Zeonor 1020 was fabricated using conventional embossing techniques to perform capillary electrophoresis for selected ion monitoring and selected reaction monitoring mass spectrometric detection of small molecules. A silicon master was microfabricated using photolithographic and dry etching processes. The microfluidic channel was embossed in the plastic from a silicon master. The embossed chip was thermally bonded with a Zeonor 1020 cover to form an enclosed channel. This channel (60-microm width, 20-microm depth, 2.0- and 3.5-cm length) provided capillary electrophoresis (CE) separation of polar small molecules without surface treatment of the polymer. A microsprayer coupled via a microliquid junction provided direct electrospray mass spectrometric detection of CE-separated components. An electric field of 0.5-2 kV/cm applied between the microsprayer and a separation buffer reservoir produced a separation of carnitine, acylcarnitine, and butylcarnitine with separation efficiencies ranging from 1,650 to 18,000 plates. Injection quantities of 0.2 nmol of these compounds produced a separation of the targeted polar small molecules without surface treatment of the polymer-abundant ion current signals and baseline separation of these compounds in less than 10 s. These results suggest the feasibility of polymeric chip-based devices for ion spray CE/MS applications.

An optical detector has been fabricated that is specific for targeted bacterial cells, by stamping an antibody grating pattern on a silicon surface. The antibody grating alone produces insignificant optical diffraction, but upon immunocapture of cells, the optical phase change produces a diffraction pattern. This technique eliminates much of the surface modifications and the secondary immunochemical or enzyme-linked steps that are common in immunoassays. Microcontact printing provides an alternative to previously reported photolithographic-mediated antibody patterning processes and uses a photolithographic process simply to produce the elastomeric stamp. We have stamped antibodies directly onto clean native oxide silicon substrates with no other chemical surface treatments. Direct binding of the antibodies to the silicon occurs in a way that still allows them to function and selectively bind antigen. The performance of the sensor was evaluated by capturing Escherichia coli O157:H7 cells on the antibody-stamped lines and measuring the intensity of the first-order diffraction beam resulting from the attachment of cells. The diffraction intensity increases in proportion to the cell density bound on the surface.

High-efficiency separation methods such as capillary electrophoresis (CE) interfaced with structural information-producing detection systems such as mass spectrometry (MS) represent invaluable tools in analytical chemistry. Most CE applications to date have been reported using UV absorption detection. To extend the analytical power of CE, new detectors which demonstrate increased speed, sensitivity, and specificity must be developed. In this paper, we describe the design of an electrospray ionization time-of-flight mass spectrometer and its performance as a detector for CE. Emphasis is placed on fulfillment of the speed and sensitivity requirements. Low attomole and low femtomole detection limits are demonstrated for continuous infusion and CE−MS operation, respectively.

A prototype amperometric fructose biosensor based on membrane-bound fructose dehydrogenase (Gluconobacter sp.) and the coenzyme ubiquinone-6 immobilized in a membrane mimetic layer on a gold electrode has been constructed and tested. A bare gold electrode first was modified through chemisorption of a mixture of octadecyl mercaptan and two short-chain disulfides, 3,3'-dithiodipropionic acid and cystamine dihydrochloride. The membrane-bound enzyme, coenzyme, and additional phospholipid were codeposited through a detergent dialysis protocol. The short-chain modifiers may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate and phospholipids enable hydrophobic interaction with the largely lipophilic, membrane-bound enzyme. At oxidizing potentials, the enzyme electrode responded with catalytic current densities up to 45 μA/cm(2) when exposed to fructose at 10 mM. The sensor exhibited a response time of less than 20 s, a sensitivity of 15 μA/cm(2)·mM and a detection limit of less than 10 μM. Biosensor measurements of d-fructose in apple and orange juice agreed to within a few percent with those made with an enzymatic spectrophotometric assay. The membrane mimetic layer effectively blocked access of interfering ascorbic acid to the electrode surface. Only a 4% positive error was observed in the presence of ascorbic acid at 5% of the fructose concentration (2 mM), which indicates that this construct could be particularly useful for quantitation of fructose in citrus juice.

Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator's surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 microg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 microg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL.

Biological arrays are hindered by the lack of uniformity in the deposition of biomaterials. Efforts aimed at improving this deposition have focused on altering the composition of the solution or the tool used to deposit the material. However, little attention has been paid to controlling material deposition by constraining the physical and chemical topography of the surface. Here we present the use of a hybrid hydrophilic/hydrophobic micropatterned surface to direct the deposition of spotted DNA on microarrays. These polymer “lift-off” arrays combine the hydrophobic surface properties of di-para-xylylene (Parylene) with photolithographically etched hydrophilic openings within the polymer. We show that the flow pattern of solutes on these substrates favors the concentration of dissolved material into the mesoscopic openings underlying the printed spot, resulting in significantly improved uniformity of deposition. Moreover, the micropatterned surface allows for increased replication of spotted materials. Finally, these polymer lift-off arrays display reduced array-to-array variation, improving the reproducibility of data acquisition. We envision that these novel substrates can be generalized to produce more uniform arrays of other patterned biomaterials.

We have demonstrated the use of a simple microfabricated electrospray ionization source for coupling microfluidic chips to mass spectrometry (MS). A polymer-based microchip, coupled to a triple quadrupole mass spectrometer, has been employed for direct infusion quantitative bioanalysis of methylphenidate (Ritalin) extracted from human urine samples. The approach used a microfabricated polymer electrospray emitter to couple a microfluidic channel to a stable electrospray ionization source. The microchip was fabricated from cycloolefin plastic plate by hot embossing and thermal bonding. This microfluidic chip contained two independent microfluidic channels, integrated with two corresponding electrospray emitters and an internal gold electrode. Liquid-liquid extraction was used to prepare urine samples, spiked with methylphenidate. A trideuterated analogue of methylphenidate (methylphenidate-d(3)) was used as the internal standard for the analysis. The system showed good electrospray stability and reproducibility with different spray tips. Four different electrospray tips were used to analyze the same sample, and the results showed very small variation with a relative standard deviation of 1.4%. A standard curve prepared for methylphenidate in urine (R(2) = 0.999) was linear over the range of 0.4-800 ng/mL. The precision of the quality control samples for three different concentrations ranged from 19.1% at 20 ng/mL, 3.2% at 200 ng/mL, to 3.5% at 667 ng/mL while the accuracy was 96.3% at 20 ng/mL, 101.2% at 200 ng/mL, and 101.6% at 667 ng/mL. No system carryover was detected even when the same device was used for sequential analysis. These results suggest the potential of this microdevice for MS-based quantitative analysis in drug discovery and development.

Microfluidic channels with two lateral dimensions smaller than 1 microm were fabricated in fused silica for high-sensitivity single-molecule detection and fluorescence correlation spectroscopy. The effective observation volumes created by these channels are approximately 100 times smaller than observation volumes using conventional confocal optics and thus enable single-fluorophore detection at higher concentrations. Increased signal-to-noise ratios are also attained because the molecules are restricted to diffuse through the central regions of the excitation volume. Depending on the channel geometries, the effective dimensionality of diffusion is reduced, which is taken into account by simple solutions to diffusion models with boundaries. Driven by electrokinetic forces, analytes could be flowed rapidly through the observation volume, drastically increasing the rate of detection events and reducing data acquisition times. The statistical accuracy of single-molecule characterization is improved because all molecules are counted and contribute to the analysis. Velocities as high as 0.1 m/s were reached, corresponding to average molecular residence times in the observation volume as short as 10 micros. Applications of these nanofabricated devices for high-throughput, single-molecule detection in drug screening and genomic analysis are discussed.

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused-silica capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI-MS analysis. The system was used both to yield the Michaelis constant (K(m)) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-microL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI-MS detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 microg of HLM protein.

The fabrication of fluidic channels with dimensions smaller than 1 microm is described and characterized in respect to their use for detection of individual DNA molecules. The sacrificial layer technique is used to fabricate these devices as it provides CMOS-compatible materials exhibiting low fluorescence background. It also allows creating microfluidics circuitry of submicrometer dimensions with great control. The small dimensions facilitate single molecule detection and minimize events of simultaneous passage of more than one molecule through the measurement volume. The behavior of DNA molecules inside these channels under an applied electrical field was first studied by fluorescence correlation spectroscopy using M13 double-stranded DNA. A linear relationship between the flow speed and applied electric field across the channel was observed. Speeds as high as 5 mm/s were reached, corresponding to only a few milliseconds of analysis time per molecule. The channels were then used to characterize a mixture of nine DNA fragments. Both the distribution and relative proportions of the individual fragments, as well as the overall concentration of the DNA sample, can be deduced from a single experiment. The amount of sample required for the analysis was approximately 10,000 molecules, or 76 fg. Other potential applications of these submicrometer structures for DNA analysis are discussed.

Recently, a microfabricated entropic trap array was demonstrated to be useful in separating large (5-200 kbp) DNA molecules efficiently (within approximately 30 min), by dc electrophoresis, on a microchip platform without a sieving matrix. This paper reports further development of the technique, with emphasis on optimizing separation selectivity and resolution. The interaction of DNA molecules with regularly spaced entropic barriers was modeled in order to predict the effect of changing various structural parameters. The selectivity (differential mobility) was shown to be dependent on the depth of deep and shallow channel regions, applied electric field, and number of entropic barriers. Experimental data were compared with the prediction of the model. It was expected from the model that, in the low-field (severe trapping) limit, separation resolution should depend only on the number of entropic traps. However, in reality, resolution did depend on the applied field because the relaxation of DNA is not achieved at high fields. The requirement and feasibility of megabase pair DNA separation with the entropic trap array device was discussed.

In response to industry's need to monitor and sample atmospheric contaminants over an 8-h day in an individual's breathing zone, the U.S. Naval Research Laboratory has designed a personal atmospheric sampler to evaluate for such contaminants as carbon monoxide, hydrogen, methane, dichlorodifluoromethane, ethane, hexane, benzene, and vinyl chloride. The small, lightweight, portable sampler collects time-integrated air samples by using an evacuated container equipped with a critical orifice rather than a pump. The sampling interval is dictated by the size of the orifice and volume of the sample container. Results show that the sampler is efficient and easy to use for low-molecular-weight compounds. The use of inert materials for construction could extend the usefulness to atmospheres containing intermediate-molecular-weight and polar compounds.