Electrical detection of viral DNA using ultramicroelectrode arrays

A fully electrical array for voltammetric detection of redox molecules produced by enzyme-labeled affinity binding complexes is shown. The electronic detection is based on ultramicroelectrode arrays manufactured in silicon technology. The 200-microm circular array positions have 800-nm-wide interdigitated gold ultramicroelectrodes embedded in silicon dioxide. Immobilization of oligonucleotide capture probes onto the gold electrodes surfaces is accomplished via thiol-gold self-assembling. Spatial separation of probes at different array positions is controlled by polymeric rings around each array position. The affinity bound complexes are labeled with alkaline phosphatase, which converts the electrochemically inactive substrate 4-aminophenyl phosphate into the active 4-hydroxyaniline (HA). The nanoscaled electrodes are used to perform a sensitive detection of enzyme activity by signal enhancing redox recycling of HA resulting in local and position-specific current signals. Multiplexing and serial readout is realized using a CMOS ASIC module and a computer-controlled multichannel potentiostat. The principle of the silicon-based electrical biochip array is shown for different experimental setups and for the detection of virus DNA in real unpurified multiplex PCR samples. The fast and quantitative electronic multicomponent analysis for all kinds of affinity assays is robust and particle tolerant.     

A whole blood immunoassay using gold nanoshells

A rapid immunoassay capable of detecting analyte within complex biological media without any sample preparation is described. This was accomplished using gold nanoshells, layered dielectric-metal nanoparticles whose optical resonance is a function of the relative size of its constituent layers. Aggregation of antibody/nanoshell conjugates with extinction spectra in the near-infrared was monitored spectroscopically in the presence of analyte. Successful detection of immunoglobulins was achieved in saline, serum, and whole blood. This system constitutes a simple immunoassay capable of detecting sub-nanogram-per-milliliter quantities of various analytes in different media within 10-30 min.     

Analysis of silicate materials using the microwave-assisted sample preparation

A technique of microwave-assisted preparation of natural and commercial silicate materials for subsequent spectrophotometric determination of the major rock-forming components (silicon, aluminum, iron, titanium, and phosphorus) is described. The total duration of analysis is several times less than for a standard technique. In particular, the time taken for the digestion is shortened to 1 h for ten samples undergoing digestion simultaneously; the time of complexation of aluminum with aluminon and of phosphorus with molybdenum heteropoly acid is reduced from a few hours to 12 min.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.     

Rapid preparation of crystalline colloidal arrays using a strong electric field dialysis

We present a simple method for rapid preparation of crystalline colloidal arrays (CCAs) by a strong electric field dialysis (SEFD). This method is based on rapid removing of ionic impurities in colloidal suspensions by applying a strong electric field. In a SEFD process, the negatively charged ions in colloidal suspensions are rapidly driven to the anode, the positively charged ones are rapidly driven to the cathode, and the colloidal particles are withheld in the dialysis tube. It was shown that the colloidal particles aggregated on the wall of the dialysis tube could block the SEFD process, which could be overcome by reversing the direction of the electric field. The purified colloidal particles can self-assemble into a crystalline colloidal array, which has an electrostatically stabilized three-dimensional periodic array of colloidal particles with a characteristic lattice spacing that can be varied by dilution. The reflection spectra show distinct peaks due to diffraction from CCAs. Atomic force microscopy (AFM) image illustrates the non-contacted ordering of the colloidal particles in the CCAs embedded in gels. This indicates the formation of high-quality single CCAs. Using a SEFD method, the preparation time of CCAs can be reduced. This new technique will greatly speed up the process of preparing polymerized crystalline colloidal arrays (PCCAs) into real-world application in the analytical field.     

DNA hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.     

Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. "Toxic hits" uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD 50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.     

Synthesis of amorphous Si nanowires from solid Si sources using microwave irradiation

Si nanowires were synthesized from Si wafers and from thin Si films deposited on various substrates by microwave irradiation. The power and time were key determinants of the diameter and morphology of the synthesized Si nanowires. The nanowires had an amorphous structure due to the extremely high heating rate. Carbon coating of the Si nanowires was easily achieved by introducing acetylene after synthesizing the nanowires. Carbon-coated Si nanowires are potential candidates for use as the anode material in next generation Li-ion batteries.     

High gradient magnetic separation of red cells from whole blood

It is demonstrated that red blood cells may be separated from other blood components using a high gradient magnetic separator. The (SI) magnetic susceptibility of red blood cells is estimated to be 3.88×10^-6when the haemoglobin is in the completely deoxygenated state. The magnetic separation effects have been studied using a filter of circular stainless steel wire with flow rates between 10^-4ms^-1and 6×10^-4ms^-1and magnetic fields in the range 0.6 to 2.4 T. The results indicate that the filter quickly saturates and the variation of filter performance with field and flowrate is discussed in terms of the force balance and the particle trajectory model. Scanning electron microscopy and free haemoglobin tests on the filtered red blood cells show no evidence of serious damage or cell rupture.     

Polyurethanes preparation using proteins obtained from microalgae

It is widely believed that the biofuels can be sustainably produced using microalgae that are known to convert CO₂ from the atmosphere to lipids, in the presence of nutrient and accumulate them as their body mass. However, when algal biofuels are produced using thermochemical route, ~30–65 % of proteins present in algae are lost due to decomposition and some of the nitrogen from amino acids is incorporated into the biofuels. The algal protein is a valuable resource that can bring additional revenue to the biorefinery by converting this co-product to high-value polyurethanes. In this work, we have demonstrated a one-step removal of proteins from algae through hydrolysis of the proteins to smaller peptides and amino acids using environment friendly flash hydrolysis (FH) process. Subcritical water was used as a reactant and as a reaction media for hydrolyzing the algae proteins via FH. Scenedesmus spp., slurry in water (3.8 %), was used as the algal feed stock during the FH process which was run at 280 °C for a residence time of 10 s. The soluble amino acids and peptides were separated from the other insoluble algal biomass components (cell wall and lipids) by filtration followed by freeze-drying. The product was then characterized by ion chromatography and Fourier transform ion cyclotron resonance mass spectrometry to determine its composition. The freeze-dried peptide and amino acids were then reacted with diamine and ethylene carbonate to produce polyols that were further processed to produce polyurethane. The relatively high hydroxyl value of these amino acid-based polyols and their compatibility with other commercially available polyols made them particularly suitable for producing rigid polyurethane foams. Due to the presence of amines and secondary amines in these polyols, the polymerization process was self-catalytic and the resulting foams are less flammable than conventional rigid polyurethane foams. The conversion of algal proteins to high-value industrial products by a relatively simple process greatly improves the value of proteins extracted from algae.     

Ultrasensitive label-free DNA analysis using an electronic chip based on carbon nanotube nanoelectrode arrays

We report the detection of DNA PCR amplicons using an ultrasensitive label-free electronic technique based on multiwalled carbon nanotube (MWNT) nanoelectrode arrays embedded in an SiO(2) matrix. Specific PCR amplicons are reliably detected using electrochemical (EC) methods through allele-specific oligonucleotide hybridization. The inherent guanine bases in the DNA amplicon target of [Formula: see text] bases serve as signal moieties with the aid of Ru(bpy)(3)(2+) mediators, providing an amplified anodic current associated with the oxidation of guanine groups at the nanoelectrode surface. The reduced size and density of the nanoelectrode array provided by MWNTs dramatically improves the sensitivity of EC detection. In addition, the abundant guanine bases in target DNA produce a large signal. Less than [Formula: see text] target amplicons can be detected on a microspot, approaching the sensitivity limit of conventional laser-based fluorescence techniques. This method also eliminates the labelling requirement and makes the measurements much simpler. This platform can be employed for developing highly automated electronic chips with multiplex nanoelectrode arrays for quick DNA analysis.     

Biomimetic autoseparation of leukocytes from whole blood in a microfluidic device

Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocyte-depleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing.     

Investigation of electrolyte measurement in diluted whole blood using spectroscopic and chemometric methods

The feasibility of using near-infrared (NIR) spectroscopy in combination with partial least-squares (PLS) regression was explored to measure electrolyte concentration in whole blood samples. Spectra were collected from diluted blood samples containing randomized, clinically relevant concentrations of Na+, K+, and Ca2+. Sodium was also studied in lysed blood. Reference measurements were made from the same samples using a standard clinical chemistry instrument. Partial least squares (PLS) was used to develop calibration models for each ion with acceptable results (Na+, R2 = 0.86, CVSEP = 9.5 mmol/L; K+, R2 = 0.54, CVSEP = 1.4 mmol/L; Ca2+, R2 = 0.56, CVSEP = 0.18 mmol/L). Slightly improved results were obtained using a narrower wavelength region (470-925 nm) where hemoglobin, but not water, absorbed indicating that ionic interaction with hemoglobin is as effective as water in causing measurable spectral variation. Good models were also achieved for sodium in lysed blood, illustrating that cell swelling, which is correlated with sodium concentration, is not required for calibration model development.     

透氧膜法由SiO2直接制备Si

为改变硅生产工艺高污染、高能耗的现状,研究了在CaCl2熔盐中利用固体透氧膜法(SOM)直接电解SiO2制备单质Si,考察了电解电压、电解时间、熔盐温度等参数对电解效果的影响,采用电子扫描显微镜和X射线衍射分析了电解产物形貌及相组成.结果表明:1100℃熔盐中,3.5 V电压下电解2 h,可制得纯Si,电流效率为89%...     

Synthesis and characterization of Fe3O4@SiO2 core-shell magnetic microspheres for extraction of genomic DNA from human whole blood

An improved procedure was described for preparation of monodisperse Fe3O4@SiO2 core-shell magnetic microspheres via solvothermal method followed by a modified St?ber process. The magnetic composite microspheres were characterized with TEM, SEM, FTIR and VSM. Genomic DNA was then extracted from human whole blood using the as synthesized magnetic microspheres on the Eppendorf epMotion5075 workstation. The quality of eluted DNA was evaluated by PCR. The method was very expeditious without using toxic compounds such as phenol or chloroform and can be used for further molecular biology experiments.     

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.