Direct transduction of allele-specific primer extension into electrical signal using genetic field effect transistor

We have developed a genetic field effect transistor (FET) for single nucleotide polymorphism (SNP) genotyping, which is based on potentiometric detection of molecular recognition on the gate insulator. Here, we report direct transduction of allele-specific primer extension on the gate surface into electrical signal using the genetic FETs. This method is based on detection of intrinsic negative charges of polynucleotide synthesized by DNA polymerase. The charge density change at the gate surface could be monitored during primer extension reaction. Moreover, three different genotypes could be successfully distinguished without any labeling for target DNA by the use of the genetic FET in combination with allele-specific primer extension. The platform based on the genetic FETs is suitable for a simple, accurate and inexpensive system for SNP genotyping in clinical diagnostics.     

A novel ISFET-type biosensor based on P450 monooxygenases

We made a biosensor based on ion-sensitive field effect transistor (ISFET) using P450 monooxygenase. ISFETs are electrical devices and have been used as pH sensors. We used genetically engineered P450 monooxygenase for our research because of its high enzymatic activity. The fusion enzyme between rat CYP1A1P450 monooxygenase and yeast NADPH-cytochrome P450 oxidoreductase was expressed in yeast Saccharomyces cerevisiae strain AH22. Yeast microsomal membranes were immobilized in an agarose layer on the ISFET. o-Deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin was catalyzed by the enzyme in the presence of nicotinamide adenine dinucleotide phosphate reduced form (NADPH). Formation of 7-hydroxycoumarin from 7-ethoxycoumarin was also measured by fluorescence. The difference of the voltage between the ISFET device and control device without enzymes showed a voltage increase along with the enzymatic reaction of P450 monooxygenases, and this voltage increase in the device was inhibited by addition of MnCl(2), an inhibitor of P450 monooxygenase. There was a positive correlation between the voltage increase in the ISFET device and the fluorescence intensity. This is the first electrochemical biosensing using P450 monooxygenases immobilized on the ISFET, and is applicable to the sensing of chlorophenol compounds.     

Detection of DNA recognition events using multi-well field effect devices

We proposed the multi-well field effect device for detection of charged biomolecules and demonstrated the detection principle for DNA recognition events using quasi-static capacitance-voltage (QSCV) measurement. The multi-well field effect device is based on the electrostatic interaction between molecular charges induced by DNA recognition and surface electrons in silicon through the Si(3)N(4)/SiO(2) thin double-layer. Since DNA molecules and DNA binders such as Hoechst 33258 have intrinsic charges in aqueous solutions, respectively, the charge density changes due to DNA recognition events at the Si(3)N(4) surface were directly translated into electrical signal such as a flat band voltage change in the QSCV measurement. The average flat band shifts were 20.7 mV for hybridization and -13.5 mV for binding of Hoechst 33258. From the results of flat band voltage shifts due to hybridization and binding of Hoechst 33258, the immobilization density of oligonucleotide probes at the Si(3)N(4) surface was estimated to be 10(8) cm(-2). The platform based on the multi-well field effect device is suitable for a simple and arrayed detection system for DNA recognition events.     

Enzyme field effect transistor (ENFET) for estimation of triglycerides using magnetic nanoparticles

Ion-selective field effect transistor (ISFET) is a robust platform to develop biosensors. A variety of methods are used including covalent attachment or polymer entrapment, to associate enzymes or antibodies to the gate surface of a FET. We have employed a novel method of retaining the enzyme molecules at the gate surface by immobilizing the enzyme on magnetic nickelferrite nanoparticles and applying a permanent magnet below the gate of the FET. We were able to estimate the triglyceride concentrations in the range of 0.1–1.5% by immobilizing a thermostable lipase on nanoparticles. Tributyrin, trioctanoate and triolein have given similar results. The reaction volume could be scaled down to 0.2 ml without a loss in slope or sensitivity. Ionic strength (>150 mM NaCl) has a strong influence on the sensitivity of the measurement. The advantages of this configuration of enzyme biosensor are reduction of mass transfer problems, increasing the amount of enzyme at the gate surface besides providing an opportunity to use a single FET device for multiple analyte detection.     

Modification of ISFETs with a monolayer of latex beads for specific detection of proteins

The so-called ion-step method is a novel potentiometric approach that can detect protein adsorbed onto the gate area of modified ion-sensitive field-effect transistors (ISFETs). In this report, a generic technology is described for immobilization of peptides and proteins to the ISFET gate in order to confer specific binding properties to the ISFET. For this, the surface of the ISFET was covered with a monolayer of Amino beads (diameter, 0.9 microm) followed by immobilization of protein ligands onto these beads. Amino beads are latex spheres that contain primary amino groups at the outer surface. Preactivation of the latex-bound amino groups with glutaraldehyde, and consecutive incubation with polylysine resulted in covalent immobilization of this polyamine, as revealed by ion stepping measurements. For ImmunoFET applications, human serum albumin (HSA) was immobilized onto the Amino bead-covered ISFETs, by passive adsorption but also by covalent coupling. Resulting devices were used for qualitative detection of alpha-HSA antibodies by means of the ion step method. The binding of antibody was very specific and fast (most of the binding was accomplished in 15 min) with signal yields up to 17 mV. Efforts to increase the antibody-binding capacity of the solid phase on the ISFET exploiting amino group activation (with glutaraldehyde or other homobifunctional cross linkers) before HSA coupling, did not improve signal yield. The bead technology described in this report is an easy, generic method for coating the ISFET with a solid phase that, using the ion-step method, can be applied to immunosensing.     

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.     

An ON/OFF biosensor based on blockade of ionic current passing through a solid-state nanopore

Single nanopores have attracted interest for their use as biosensing devices. In general, methods involve measuring ionic current blockades associated with translocation of analytes through the nanopore, but the detection of such short time lasting events requires complex equipment and setup that are critical for convenient routine biosensing. Here we present a novel biosensing concept based on a single nanopore in a silicon nitride membrane and two anchor-linked DNA species that forms trans-pore hybrids, realizing a stable blockade of ionic current through the pore. Molecular recognition events affecting the DNA hybrids cause a pore opening and the consequent establishment of an ionic current. In the present implementation of the device, we constructed a magnetic bead/streptavidin/biotin-DNA1/DNA2-biotin/streptavidin/Quantumdot-cluster complex (where DNA1 is a mismatched reverse complement of DNA2) through a sub-micrometric pore and monitored DNA strand displacement events occurring after addition of an oligonucleotide complementary to DNA2. The electric and mechanical aspects of the novel device, as well as its potential in biosensing are discussed.     

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.     

Magnetic scanometric DNA microarray detection of methyl tertiary butyl ether degrading bacteria for environmental monitoring

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications.     

Electrochemical sensor based on photopolymeric membranes for determining urea

A new electrochemical enzymatic sensor based on the ion selective field effect transistors (ISFETs) and photocurable membrane was developed for the determination of urea. For the immobilization of urease on the gate surface of the ISFET a simple method, involving the use of liquid photocurable compositions on the basis of vinylpirollidone, oligouretanemetacrylate and oligocarbonatemetacrylate, was applied. The linearange of the response of the developed electrochemical sensor lies in the range 0.05-20 mM. The latter corresponds to the claims of the medical practice. The overall time of the analysis is 5-10 min. The effects of the buffer concentration and its pH as well as temperature and presence of ammonia ions in the measuring medium on the amplitude of the sensor response were estimated. The duration of sensor work is as shortest 40 days. The proposed sensor on the basis of the ISFET is promising for the express analysis of the level urea in blood, while the developed method of membrane preparation with entrapped enzyme can be combined with the integral technology of producing of the biosensors semiconductor transducers.     

金结合多肽及其在生物传感领域的应用

金结合多肽是近年来通过生物展示技术或人工设计所获得的一类可以特异性与金结合的多肽,因其良好的生物相容性及易修饰性,针对此类生物大分子的研究和应用成为包括生物传感在内的众多领域的研究热点。金结合多肽多用于生物传感器的敏感膜制备,具有识别分子有序定位、反应步骤少、条件温和、高灵敏度的优点。我们在简要总结金结合多肽的代表性序列及其与金的结合机理的前提下,评述了金结合多肽在生物传感领域的应用,着重论述了利用基因工程技术表达含有金结合多肽的融合蛋白这一敏感膜关键器件的方法途径。     

The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.     

Differential adhesion of microspheres mediated by DNA hybridization I: experiment

We have developed a novel method to study collective behavior of multiple hybridized DNA chains by measuring the adhesion of DNA-coated micron-scale beads under hydrodynamic flow. Beads coated with single-stranded DNA probes are linked to surfaces coated with single target strands through DNA hybridization, and hydrodynamic shear forces are used to discriminate between strongly and weakly bound beads. The adhesiveness of microspheres depends on the strength of interaction between DNA chains on the bead and substrate surfaces, which is a function of the degree of DNA chain overlap, the fidelity of the match between hybridizing pairs, and other factors that affect the hybridization energy, such as the salt concentration in the hybridization buffer. The force for bead detachment is linearly proportional to the degree of chain overlap. There is a detectable drop in adhesion strength when there is a single base mismatch in one of the hybridizing chains. The effect of single nucleotide mismatch was tested with two different strand chemistries, with mutations placed at several different locations. All mutations were detectable, but there was no comprehensive rule relating the drop in adhesive strength to the location of the defect. Since adhesiveness can be coupled to the strength of overlap, the method holds promise to be a novel methodology for oligonucleotide detection.     

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.     

Permanent magnet actuation for magnetic bead-based DNA extraction

Background

Recently, automatic molecular diagnostic devices to extract DNA have been extensively developed using magnetic beads. While various methods can be applied to the control of the beads, the efficiency of the control when incorporated in automatic devices has not been studied. This paper proposes a compact magnet actuation method for the control of magnetic beads for DNA extraction, and compares the efficiency to the already available magnetic bead-based DNA extraction device. A permanent magnet was preferred for its compactness, while an electro-magnet provides easy operation. After investigating various methods to actuate the magnet with perspective to the size, circuit complexity, and power requirement, we determined the solenoid actuation method to be most efficient. To further reduce the dimension of the overall actuation device, direct actuation of the permanent magnet to control the hold/release of the beads was employed in this paper. The proposed method was compared with the conventional solenoid actuator with a metal plunger. An experimental fluidics device was set up with a fluidic channel and a syringe pump. The bead holding performance against the fluid speed was tested while a fixed amount of beads was loaded into the center of the channel. The group velocity of the beads was analyzed via image processing to determine whether the magnet was sufficient to hold the beads. The required power and space was analyzed and compared qualitatively and quantitatively.

Result

The proposed direct actuation method was capable of holding the beads at faster fluidic speed than the conventional solenoid actuator. The required power was comparable contemplating the high initial power of the solenoid actuator, and required much smaller space since no plunger was needed.

Conclusions

The direct actuation of the permanent magnet using a solenoid coil showed enhanced performance in holding the beads via permanent magnet, with less complexity of the actuation circuit and space. The proposed method therefore can efficiently improve the overall performance of the bead-based DNA extraction.

     

End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads

To immobilize DNA fragments onto magnetic beads coated with streptavidin for isolation purpose, it is important to label one biotin molecule at one terminus of DNA fragment. After failure to label long DNA with biotin by PCR and filling-in reaction, a 9.2 kb DNA was labeled with biotin by a modified ligation strategy. A simple method is also reported to detect the quantity and integrity of DNA immobilized on the magnetic beads.     

DNA framework-engineered electrochemical biosensors

Self-assembled DNA nanostructures have shown remarkable potential in the engineering of biosensing interfaces, which can improve the performance of various biosensors. In particular, by exploiting the structural rigidity and programmability of the framework nucleic acids with high precision, molecular recognition on the electrochemical biosensing interface has been significantly enhanced, leading to the development of highly sensitive and specific biosensors for nucleic acids, small molecules,proteins, and cells. In this review, we summarize recent advances in DNA framework-engineered biosensing interfaces and the application of corresponding electrochemical biosensors.     

An ISFET-based immunosensor for the detection of beta-Bungarotoxin

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).     

Analysing a magnetic molecule detection system--computer simulation

The detection of single molecules, e.g. in biology is possible by marking the interesting molecules with magnetic beads and detect the influence of the beads on giant magnetoresistance (GMR)/tunnel magnetoresistance (TMR)/spin valve (SV) sensors. The development of suitable multilayers has been studied experimentally as well as theoretically in order to optimize the sensor parameters. A finite difference (FD) method including the usually used contributions to the total energy [exchange, antiferromagnetically (af) coupling, anisotropy and magnetostatic] is used for the simulation with additional contributions to the local field according to the stray fields of the beads. In this work, we will show the results of micromagnetic calculations of the magnetization behavior of GMR/TMR sensors considering also the interaction between the domains in the magnetic layers of the sensor and the bead area. We can present first calculations where the bead particles (signal source) and the magnetic layers (sensor device) are considered as a whole magnetic ensemble.     

Analog-digital molecular cell computer.

The living cell is controlled by a molecular stochastic computer of parallel-successive action. MCC may be equivalent to the universal computer. MCC operates with molecule-words (DNA, RNA, proteins) according to the programme recorded in DNA and RNA. Operations are produced by molecular devices (RNA- and DNA-polymerases, ligases, proteinases and so on). Molecular devices operating with molecule-words are recorded on molecules themselves, and they are read off by ribosomes. Therefore the programme of the reorganization of the programme itself may be recorded on the molecule-words. MCC operates with molecular words having definite addresses. The words and the operators collide by Brownian movement and combine if the molecular surface of address segment is complementary and properly oriented. It is possible to reproduce not only the programmes but also the operators of MCC. The molecular computer operates with word-molecules according to the programme, recorded in DNA, with the aim of predicting an outer situation in the next time-moment and selecting of a correct answer by synthesis of suitable proteins and other substances and also by macroscopic motion. Each step of directed calculation is needed of the consumption of minimally necessary portion of free energy and search is due to the Brownian movement without free energy loss. Cyclic nucleotides are intraneuron membrane potential controlling systems which can be described as molecular diffusional analog computers, well fitted to solve mathematical physics equations if there are high frequency generators and regulators of cyclic nucleotides sources and sinks. It is suggested that molecular proton channels in an electric field are such generators of 10(11)-10(12) Hz. Biophysics cannot use the ordinary laws of physics and must take into account the influence on the phenomena to be studied, not only of a measurement but also of a calculation process in the real device predicting the future.