Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene

Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16^Ink4a) were used to be immobilized on the electrochemical array (ECA) chip. Using 1 μL of 10 ng/μL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.     

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed “U shape” reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications.     

Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry

Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of l-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring.

A new silent mutation found in the Chinese PAH locus and its role in the prenatal diagnosis of phenylketonuria

A silent mutation or sequence polymorphism. A to T substitution at codon 399 in exon 11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The frequencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09, respectively, based on the analyses of 100 normal individuals and 39 PKU patients using DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagnosis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPs linkage analysis, was prenatally diagnosed with this genetic marker.     

A NEW SILENT MUTATION FOUND IN THE CHINESE PAH LOCUS AND ITS ROLE IN THE PRENATAL DIAGNOSIS OF PHENYLKETONURIA

A silent mutation or sequence polymorphism, A to T substitution at codon 399 in exon11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The fre-quencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09,respectively, based on the analyses of 100 normal individuals and 39 PKU patients usingDNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridizationmethods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagno-sis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPslinkage analysis, was prenatally diagnosed with this genetic marker.     

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol?L^?1 for all B19 genotype oligonucleotides and 650 pmol?L^?1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

Planar chip device for PCR and hybridization with surface acoustic wave pump

We have developed a microfluidic device operating at a planar surface instead of a closed channel network. The fluid is transported in single droplets using surface acoustic waves (SAW) on a piezoelectric LiNbO(3) substrate. The surface of the piezo is chemically structured to induce high contact angles of the droplets or enclose areas where the liquid can wet the substrate. Combining the SAW technique with thin film resistance heaters, a biological analysis chip with integrated DNA amplification by PCR and hybridization was designed. To prevent evaporation of the PCR reagents at high temperatures the sample is enclosed in droplets of mineral oil. On this chip the SAW resolves dried primers, shifts the oil capped liquid between the two heaters and mixes during hybridization. The chip is able to perform a highly sensitive, fast and specific PCR with a volume as low as 200 nl. During the temperature cycles an online monitoring of the DNA concentration is feasible with an optical unit, providing a sensitivity of 0.1 ng. After PCR the product is moved to the second heater for the hybridization on a spotted DNA array. With our chip we were able to detect a single nucleotide polymorphism (SNP) responsible for the Leiden Factor V syndrome from human blood.     

实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.     

微流控芯片电泳快速分离脂蛋白

描述了一种芯片电泳快速分离脂蛋白的方法. 利用自制的微流控芯片及激光诱导荧光技术电泳分离经硝基苯并噁二唑-C₆-酰基鞘胺醇预染的脂蛋白标本, 在40 mmol/L tricine缓冲液(pH 9.4)中加入40 mmol/L甲基葡胺, 在500 V电压下40 s进样, 在2000 V 电压下2 min内完成分离, 可出现低密度脂蛋白(LDL)与高密度脂蛋白(HDL)两条脂蛋白区带, 5次重复性试验其出峰时间变异系数(CV)为2.6%. 本法为高血脂患者提供了一种快速、简便、灵敏、重复性好的诊断方法.     

A gel-based visual immunoassay for non-instrumental detection of chloramphenicol in food samples

A gel-based non-instrumental immuno-affinity assay was developed for the rapid screening of chloramphenicol (CAP) in food samples with the limit of detection (LOD) of 1 μg L⁻¹. The immuno-affinity test column (IATC) consisted of a test layer containing anti-CAP antibody coupled gel, and a control layer with anti-HRP antibody coupled gel. Based on the direct competitive immuno-reaction and the horseradish peroxidase enzymatic reaction, the test results could be evaluated visually. Basically, blue color development represented the negative results, while the absence of color development represented the positive results. In this study, CAP spiked samples of raw milk, pasteurized milk, UHT milk, skimmed milk powder, acacia honey, date honey, fish and shrimp were tested. Little or none sample pretreatment was required for this assay. The whole procedure was completed within 10 min. In conclusion, the gel-based immuno-affinity test is a simple, rapid, and promising on-site screening method for CAP residues in food samples, with no instrumental requirement.     

A method for DNA detection in a microchannel: fluid dynamics phenomena and optimization of microchannel structure

A simple DNA diagnosis method using microfluidics has been developed which requires simple and straightforward procedures such as injection of sample and probe DNA solutions. This method takes advantage of the highly accurate control of fluids in microchannels, and is superior to DNA microarray diagnosis methods due to its simplicity, highly quantitative determination, and high-sensitivity. The method is capable of detecting DNA hybridization for molecules as small as a 20 mer. This suggests the difference in microfluidic behavior between single strand DNA (ssDNA) and double stranded DNA (dsDNA). In this work, influence of both the inertial force exerted on DNA molecules and the diffusion of DNA molecules was investigated. Based on the determination of these parameters for both ssDNA and dsDNA by experiments, a numerical model describing the phenomena in the microchannel was designed. Computational simulation results using this model were in good agreement with previously reported experimental results. The simulation results showed that appropriate selection of the analysis point and the design of microchannel structure are important to bring out the diffusion and inertial force effects suitably and increase the sensitivity of the detection of DNA hybridization, that is, the analytical performance of the microfluidic DNA chip.     

Disposable sensors for rapid screening of mutated genes

A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH₃)₅?L]²⁺, where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at –0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/μL PCR product can be detected.     

Development of a fast and efficient ultrasonic-based strategy for DNA fragmentation

Several ultrasound-based platforms for DNA sample preparation were evaluated in terms of effective fragmentation of DNA (plasmid and genomic DNA)—ultrasonic probe, sonoreactor, ultrasonic bath and the newest Vialtweeter device. The sonoreactor showed the best efficiency of DNA fragmentation while simultaneously assuring no cross-contamination of samples, and was considered the best ultrasonic tool to achieve effective fragmentation of DNA at high-throughput and avoid sample overheating. Several operation variables were studied—ultrasonication time and amplitude, DNA concentration, sample volume and sample pre-treatment—that allowed optimisation of a sonoreactor-based strategy for effective DNA fragmentation. Optimal operating conditions to achieve DNA fragmentation were set to 100% ultrasonic amplitude, 100 μL sample volume, 8 min ultrasonic treatment (2 min/sample) for a DNA concentration of 100 μg mL⁻¹. The proposed ultrasonication strategy can be easily implemented in any laboratory setup, providing fast, simple and reliable means for effective DNA sample preparation when fragmentation is critical for downstream molecular detection and diagnostics protocols.     

Microchip capillary electrophoresis with electrochemical detector for precolumn enzymatic analysis of glucose, creatinine, uric acid and ascorbic acid in urine and serum

An integrated multiple-enzymatic assay was performed on a (microchip capillary electrophoresis) μCE-EC chip capable of precise intake of sample or reagents in nanoliters. Incorporating multiple-enzyme assay into the μCE chip is relatively new—rendering simultaneous analysis of creatinine and uric acid a snap.Added to the list of merits in this study are the enhanced sensitivity down to 1 μM and a broader spectrum of analytes—inclusive of glucose for the long-time sufferers of diabetes. The performance was orchestrated to attain the claimed level: employing the end-channel electrode mode to tame the noises and the precolumn enzymatic reaction to stabilize the baseline. The 10 μm embedded Pt electrode, deposited at the end of the 30 μm wide separation channel, benefited chip fabrication besides noise reduction. The optimized conditions were 20 mM phosphate buffer (pH 7.5), +1.5 kV separation voltage and +1.0 V detection potential (versus Ag/AgCl). The migration time was repeatable within the deviation of 0.5% R.S.D. (n=7), but the peak currents ranged from 1.5 to 2.2% R.S.D. The detection limits (S/N=3) ranged from 0.71 μM for ascorbic acid to 10 μM for glucose. The calibration curve was linear from 10 to 800 μM (R²>0.995). Glucose, creatinine, uric acid and ascorbic acid as model analytes, in pure form or in serum and urine samples, were tested to verify its feasibility.     

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on‐demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary‐ and chip‐based methods offering low consumable costs (<0.1$) circumventing cost‐intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2–4 μL in volume, a semi‐contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non‐contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR‐grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi‐circular cross section of adaptable width (∼200–600 μm) and height (∼30–80 μm) as well as a typical length of 15–55 mm. Layout of such liquid structures is adaptable on‐demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label‐free DNA ladder (100–1000 bp) at 100 V/cm via in‐line staining and laser induced fluorescent end‐point detection using an automated prototype.     

Drop-to-drop microextraction across a supported liquid membrane by an electrical field under stagnant conditions

Electromembrane extraction (EME) of basic drugs from 10 μL sample volumes was performed through an organic solvent (2-nitrophenyl octyl ether) immobilized as a supported liquid membrane (SLM) in the pores of a flat polypropylene membrane (25 μm thickness), and into 10 μL 10 mM HCl as the acceptor solution. The driving force for the extractions was 3–20 V d.c. potential sustained over the SLM. The influence of the membrane thickness, extraction time, and voltage was investigated, and a theory for the extraction kinetics is proposed. Pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from pure water samples with recoveries ranging between 33% and 47% after only 5 min of operation under totally stagnant conditions. The extraction system was compatible with human urine and plasma samples and provided very efficient sample pretreatment, as acidic, neutral, and polar substances with no distribution into the organic SLM were not extracted across the membrane. Evaluation was performed for human urine, providing linearity in the range 1–20 μg/mL, and repeatability (RSD) in average within 12%.     

Functional integration of DNA purification and concentration into a real time micro-PCR chip

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

Microchip-based immunoassay system with branching multichannels for simultaneous determination of interferon-gamma

A bead-bed immunoassay system suitable for simultaneous assay of multiple samples was constructed on a microchip. The chip had branching multichannels and four reaction and detection regions; the constructed system could process four samples at a time with only one pump unit. Interferon gamma was assayed by a 3-step sandwich immunoassay with the system coupled to a thermal lens microscope as a detector. The biases of the signal intensities obtained from each channel were within 10%, and coefficients of variation were almost the same level as the single straight channel assay. The assay time for four samples was 50 min instead of 35 min for one sample in the single-channel assay; hence higher throughput was realized with the branching structure chip.     

Spatially addressable bead-based biosensor for rapid detection of beta-thalassemia mutations

A simple glass-polymer bead-based biosensor was validated for the detection of beta-thalassemia mutations. Different bead types, each carrying allele-specific probes targeting a particular mutation on the beta-globin gene, were immobilized and distinguished on the chip based on their spatial addresses. Genomic DNA samples carrying various single nucleotide transitions and transversions in the beta-globin gene were subjected to polymerase chain reaction and asymmetric amplification in the presence of Cy3-labeled primers, followed by hybridization onto the chip and detection under an epifluorescent microscope. Mutations that were heterozygous or homozygous were easily detected on the device based on the signal intensity difference (or similarity) between the wildtype and mutant probes. This device successfully detected all six common beta-globin gene mutations within 30 min. The number of targeted mutations on this chip can be easily expandable through the introduction of additional probe sets.