Comparison of a prototype magnetoresistive biosensor to standard fluorescent DNA detection

We present a comparative analysis of a magnetoresistive biosensor to standard fluorescent DNA detection. The biosensor consists of giant magnetoresistive (GMR) type Cu/Ni(80)Fe(20) multilayers in the second antiferromagnetic coupling maximum. Each of the 206 elements of the magnetoresistive biosensor is patterned into a spiral-shaped line that can cover the area of a typical DNA spot (70 microm diameter). The probe DNA is assembled on top of the sensor elements in different concentrations ranging from 16 pg/microl to 10 ng/microl. Complementary biotin-labeled analyte DNA is hybridized to the probe DNA at a concentration of 10 ng/microl. A number of different commercially available magnetic microspheres are investigated to determine the most appropriate markers. The experimental comparison shows that the relative sensitivity of the magnetoresistive biosensor is superior to the fluorescent detection at low probe DNA concentrations.     

An integrated and sensitive detection platform for magneto-resistive biosensors

A compact biosensor platform with giant magneto-resistive (GMR) sensors suited for the detection of superparamagnetic nanoparticle labels is presented. The platform consist of disposable biosensor cartridges and an electronic reader, which enables quantitative detection with high analytical performance, combined with robustness, ease of use and at low cost. In order to optimise the signal-to-noise ratio (SNR), magnetic labels are excited at high frequency. Wires, integrated in the silicon of the sensor chip are used to generate a well-defined magnetic field on the sensor surface, thus removing the need for mechanical alignment with external apparatus. A signal modulation scheme is applied to obtain optimal detection accuracy. The platform is scalable and can be adapted according to application-specific requirements. Experimental results indicate that three beads of 300 nm diameter can be detected on a sensor surface of 1500 microm2 for a measurement time of 1s.     

Giant magnetoresistive biochip for DNA detection and HPV genotyping

A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10nM in most cases, but concentrations of 10pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.     

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.     

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH(2)) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β-d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0×10(-10) mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×10(2) to 2.0×10(3) cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.     

Detection and manipulation of biomolecules by magnetic carriers

The detection and manipulation of single molecules on a common platform would be of great interest for basic research of biological or chemical systems. A promising approach is the application of magnetic carriers. The principles are demonstrated in this contribution. It is shown that paramagnetic beads can be detected by highly sensitive magnetoresistive sensors yielding a purely electronic signal. Different configurations are discussed. The capability of the sensors to detect even single markers is demonstrated by a model experiment. In addition, the paramagnetic beads can be used as carriers for biomolecules. They can be manipulated on-chip via currents running through specially designed line patterns. Thus, magnetic markers in combination with magnetoresistive sensors are a promising choice for future integrated lab-on-a-chip systems.     

A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips

Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

High-throughput SPR sensor for food safety

High-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of nucleic acids identifying specific bacterial pathogens is reported. The biosensor consists of a high-performance SPR imaging sensor with polarization contrast and internal referencing (refractive index resolution 2 x 10(-7) RIU) and an array of DNA probes microspotted on the surface of the SPR sensor. It is demonstrated that short sequences of nucleic acids (20-23 bases) characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of specific DNA or RNA sequences can be performed in less than 15 min by the reported SPR sensor.     

High sensitivity detection of molecular recognition using magnetically labelled biomolecules and magnetoresistive sensors

Small magnetoresistive spin valve sensors (2 x 6 microm(2)) were used to detect the binding of single streptavidin functionalized 2 microm magnetic microspheres to a biotinylated sensor surface. The sensor signals, using 8 mA sense current, were in the order of 150-400 microV for a single microsphere depending on sensor sensitivity and the thickness of the passivation layer over the sensor surface. Sensor saturation signals were 1-2 mV representing an estimated 6-20 microspheres, with a noise level of approximately 10 microV. The detection of biomolecular recognition for the streptavidin-biotin model was shown using both single and differential sensor architectures. The signal data compares favourably with previously reported signals for high numbers of magnetic microspheres detected using larger multilayered giant magnetoresistance sensors. A wide range of applications is foreseen for this system in the development of biochips, high sensitivity biosensors and the detection of single molecules and single molecule interactions.     

Ligase-based multiple DNA analysis by using an electrochemical sensor array

We herein report an electrochemical biosensor for the sequence-specific detection of DNA with high discrimination ability for single-nucleotide polymorphisms (SNPs). This DNA sensor was constructed by a pair of flanking probes that "sandwiched" the target. A 16-electrode electrochemical sensor array was employed, each having one individual DNA capture probe immobilized at gold electrodes via gold-thiol chemistry. By coupling with a biotin-tagged detection probe, we were able to detect multiple DNA targets with a single array. In order to realize SNP detection, a ligase-based approach was employed. In this method, both the capture probe and the detection probe were in tandem upon being hybridized with the target. Importantly, we employed a ligase that specifically could ligate tandem sequences only in the absence of mismatches. As a result, when both probes were complementary to the target, they were ligated in the presence of the ligase, thus being retained at the surface during the subsequent stringent washing steps. In contrast, if there existed 1-base mismatch, which could be efficiently recognized by the ligase, the detection probe was not ligated and subsequently washed away. A conjugate of avidin-horseradish peroxidase was then attached to the biotin label at the end of the detection probe via the biotin-avidin bridge. We then electrochemically interrogated the electrical current for the peroxidase-catalyzed reduction of hydrogen peroxide. We demonstrated that the electrochemical signal for the wild-type DNA was significantly larger than that for the sequence harboring the SNP.     

Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads

A novel and sensitive fluorescence biosensor based on aptamer and rolling circle amplification for the determination of cocaine was developed in the present work. Here cocaine aptamers immobilized onto Au nanoparticles modified magnetic beads hybridized with short DNA strand. In the presence of cocaine, the short DNA strand was displaced from aptamer owing to cocaine specially binding with aptamer. Next, the short DNA strand was separated by magnetic beads and used to originate rolling circle amplification as primer. The end products of rolling circle amplification were detected by fluorescence signal generation upon molecular beacons hybridizing with the end products of rolling circle amplification. With rolling circle amplification and the separation by magnetic beads reducing the background signal, the new strategy was suitable for the detection of as low as 0.48 nM cocaine. Compared with reported cocaine sensors, our method exhibited excellent sensitivity. Our new strategy may provide a platform for numerous proteins and low molecular weight analytes to highly sensitively detect by DNA amplification.     

Design of buffer exchange surfaces and sensor chips for biosensor chip mass spectrometry

The feasibility of buffer exchange in biosensor chip mass spectrometry, along with the construction of base sensor chips and use of alternative chip chemistries, is demonstrated in this work. Beta-2-microglobulin (beta2m) was used as an analyte and captured in the first flow cell (FC1) on the sensor chip surface by an immobilized anti-beta2m antibody. Low pH buffer was then used to elute the captured analyte from the flow cell and route it to a second flow cell (FC2) downstream that served as a cation exchanger that retains the analyte. Following additional washes in FC1, the analyte present in FC2 was either eluted with a higher pH buffer (to demonstrate the possibility of elution into a downstream trypsin flow cell), or it was subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis to verify its presence in FC2. In a separate experiment, a gold-sputtered glass slide (base chip) was activated through a formation of 11-mercaptoundecanoic acid self-assembled monolayer and via reaction with 1,1"-carbonyldiimidazole. The activated chip was placed manually into the biosensor and two surfaces (flow cells) were derivatized with antibodies to beta2m and cystatin C (cysC). To evaluate the chip performance, diluted human urine aliquot was injected over the flow cells. Following the surface plasmon resonance analysis, the chip was MALDI-TOF MS analyzed, yielding signals from beta2m and cysC from their respective flow cells. Artifacts arising from the surface chemistries were not observed in the analysis.     

Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network

A hand held device has been designed for the immunomagnetic detection and quantification of the pathogen Escherichia coli O157:H7 in food and clinical samples. In this work, a technology to manufacture a Lab on a Chip that integrates a 3D microfluidic network with a microfabricated biosensor has been developed. With this aim, the sensing film optimization, the design of the microfluidic circuitry, the development of the biological protocols involved in the measurements and, finally, the packaging needed to carry out the assays in a safe and straightforward way have been completed. The biosensor is designed to be capable to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody-antigen reaction. The giant magnetoresistive multilayer structure implemented as sensing film consists of 20[Cu(5.10nm)/Co(2.47 nm)] with a magnetoresistance of 3.20% at 235Oe and a sensitivity up to 0.06 Omega/Oe between 150Oe and 230Oe. Silicon nitride has been selected as optimum sensor surface coating due to its suitability for antibody immobilization. In order to guide the biological samples towards the sensing area, a microfluidic network made of SU-8 photoresist has been included. Finally, a novel packaging design has been fabricated employing 3D stereolithographic techniques. The microchannels are connected to the outside using standard tubing. Hence, this packaging allows an easy replacement of the used devices.     

On-chip manipulation and detection of magnetic particles for functional biosensors

We demonstrate the real-time on-chip detection and manipulation of single 1 microm superparamagnetic particles in solution, with the aim to develop a biosensor that can give information on biological function. Our chip-based sensor consists of micro-fabricated current wires and giant magneto resistance (GMR) sensors. The current wires serve to apply force on the particles as well as to magnetize the particles for on-chip detection. The sensitivity profile of the sensor was reconstructed by simultaneously measuring the sensor signal and the position of an individual particle crossing the sensor. A single-dipole model reproduces the measured sensitivity curve for a 1 microm bead. For a 2.8 microm bead the model shows deviations, which we attribute to the fact that the particle size becomes comparable to the sensor width. In the range between 1 and 10 particles, we observed a linear relationship between the number of beads and the sensor signal. The real-time detection and manipulation of individual particles opens the possibility to perform on-chip high-parallel single-particle assays.     

A picoliter chamber array for cell-free protein synthesis

The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.     

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.     

DNA芯片制作原理及其杂交信号检测方法

文章讨论了DNA芯片的制作原理和杂交信号的检测方法。依其结构,DNA芯片可分为两种形式,DNA阵列和寡核苷酸微芯片。DNA芯片的制作方法主要有光导原位合成法和自动化点样法。DNA芯片与标记的探针或DNA样品杂交,并通过探测杂交信号谱型来实现DNA序列或基因表达的分析。适应于DNA芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合使用CCD相机的荧光显微镜、光纤生物传感器、化学发生法、光激发磷光物质存储屏法、光散射法等。