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.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

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.     

DNA hybridization assay at individual,biofunctionalized zinc oxide nanowires

Reliable and efficient identification of DNA is a major goal in on‐site diagnostics. One dimensional nanostructures like nanowires (NW) represent potential sensor structures due to their extreme surface‐to‐bulk ratio, enabling enhanced biomolecule binding which results in optimal signals. While silicon NW are already well studied, NW made from other materials with promising properties like ZnO are not yet established as NW sensor material for bioanalytics. Here we demonstrate the DNA functionalization of ZnO NW even at the single NW level and their successful application in a DNA hybridization assay. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Nanovesicle-based bioelectronic nose platform mimicking human olfactory signal transduction

We developed a nanovesicle-based bioelectronic nose (NBN) that could recognize a specific odorant and mimic the receptor-mediated signal transmission of human olfactory systems. To build an NBN, we combined a single-walled carbon nanotube-based field effect transistor with cell-derived nanovesicles containing human olfactory receptors and calcium ion signal pathways. Importantly, the NBN took advantages of cell signal pathways for sensing signal amplification, enabling ≈ 100 times better sensitivity than that of previous bioelectronic noses based on only olfactory receptor protein and carbon nanotube transistors. The NBN sensors exhibited a human-like selectivity with single-carbon-atomic resolution and a high sensitivity of 1 fM detection limit. Moreover, this sensor platform could mimic a receptor-meditated cellular signal transmission in live cells. This sensor platform can be utilized for the study of molecular recognition and biological processes occurring at cell membranes and also for various practical applications such as food screening and medical diagnostics.     

Electrochemical study of the XNA on Gold microarray

A novel electrode array was developed based on the XNA on Gold trade mark microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold electrode. The electrochemical reaction of K4Fe(CN)6 was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)6 to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis.     

Phage-based label-free biomolecule detection in an opto-fluidic ring resonator

We have developed a sensitive and inexpensive opto-fluidic ring resonator (OFRR) biosensor using phage as a receptor for analyte detection. Phages have distinct advantages over antibodies as biosensor receptors. First, affinity selection from large libraries of random peptides displayed on phage provides a generic method of discovering receptors for detecting a wide range of analytes with high specificity and sensitivity. Second, phage production can be less complicated and less expensive than antibody production. Third, phages withstand harsh environments, reducing the environmental limitations and enabling regeneration of the biosensor surface. In this work, filamentous phage R5C2, displaying peptides that bind streptavidin specifically, was employed as a model receptor to demonstrate the feasibility of a phage-based OFRR biosensor. The experimental detection limit was approximately 100pM streptavidin and the K(d(apparent)) is 25pM. Specificity was verified using the RAP 5 phage, which is not specific to streptavidin, as the negative control. Sensing surface regeneration results show that the phage maintained functionality after surface regeneration, which greatly improves the sensors' reusability. The phage-based OFRR biosensor will become a promising platform for universal biomolecule detection with high sensitivity, low cost, and good reusability.     

Electrochemical magneto immunosensing of antibiotic residues in milk

A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads--such as those based on the use of Protein A or carboxylate modified magnetic beads - ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 microg L(-1) (5.92 nmol L(-1)) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.     

Sensitive immunosensor for tumor necrosis factor α based on dual signal amplification of ferrocene modified self-assembled peptide nanowire and glucose oxidase functionalized gold nanorod

Sensitive electrochemical immunosensor for the detection of protein biomarker tumor necrosis factor α (TNF-α) was reported that uses ferrocene carboxylic acid (Fc) functionalized self-assembled peptide nanowire (Fc-PNW) as sensor platform and glucose oxidase (GOx) modified gold nanorod (GNR) as label. Greatly enhanced sensitivity is achieved based on a dual signal amplification strategy: first, the synthesized Fc-PNW used as the sensor platform increased the loading of primary anti-TNF-α antibody (Ab(1)) onto electrode surface due to its large surface area. At the same time, the Fc moiety on the nanowire is used as a mediator for GOx to catalyze the glucose reaction. Second, multiple GOx and secondary anti-TNF-α antibody (Ab(2)) molecules are bounded onto each GNR to increase the sensitivity of the immunosensor. After the preparation of the immunosensor based on the traditional sandwich protocol, the response of the immunosensor towards glucose was used as a signal to differentiate various concentrations of TNF-α. The resulting immunosensor has high sensitivity, wide linear range (0.005-10ng/mL) and good selectivity. This immunosensor preparation strategy is a promising platform for clinical screening of protein biomarkers.     

Practical biophysics: Sensors for rapid detection of biological targets utilizing target-induced oligonucleotide annealing

Detection and quantitation of biomolecules is one of the most commonly performed measurements in biomedical research and clinical diagnostics. There is high demand for convenient, rapid and sensitive biomolecule detection methodologies. In this review we discuss a family of sensors that have been developed in our laboratory that share a common simple biophysical mechanism of action and that are capable of rapid detection of a diverse range of biological targets. The sensors generate fluorescence signal in the presence of the target molecule through target-induced association of short fluorochrome-labeled complementary oligonucleotides that are attached to target recognition elements of the sensors (antibodies, aptamers, etc.) via nanometer scale flexible linkers. This sensor design can be used for detecting proteins, antibodies, nucleic acids and whole cells. The assays using these sensors require only adding a sample to the sensor mix followed by simple fluorescence intensity readout. The simplicity, the speed of detection and the potential for miniaturization are the main assets of these sensors.     

Nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) assay

Here we have developed a novel nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) strategy for detection of genome. The key idea of this method is integrating the two in situ processes: PCR on the surface of magnetic nanoparticles (MNPs) and magnetic beads based ECL readout platform, to avoid some laborious manual operations and achieve rapid yet sensitive detection. At first, the approach employs a pair of functional primers for amplification: one is tris-(2,2'-bipyridyl) ruthenium (TBR) labeled primer; the other one is nano-magnetic primer which is prepared by attaching the primer to the surfaces of MNPs. With the presence of DNA analyte and PCR mixture, the TBR labeled products are directly loaded and enriched on the surface of MNPs during PCR cycling. Then the MNPs-TBR complexes can be analyzed by a magnetic ECL platform without any post-modification or post-incubation. Finally, we used Listeria monocytogenes as the target to examine these desirable properties of this assay, reaching a detection limit of 500 fg/μL for genome in 1 h. The proposed study has provided the evidence as a proof-of-concept, thus having potential for development of automatic mode for detection of specific gene.     

Improved Detection of Magnetic Signals by a MEMS Sensor Using Stochastic Resonance

We introduce the behavior of the electrical output response of a magnetic field sensor based on microelectromechanical systems (MEMS) technology under different levels of controlled magnetic noise. We explored whether a particular level of magnetic noise applied on the vicinity of the MEMS sensor can improve the detection of subthreshold magnetic fields. We examined the increase in the signal-to-noise ratio (SNR) of such detected magnetic fields as a function of the magnetic noise intensity. The data disclosed an inverted U-like graph between the SNR and the applied magnetic noise. This finding shows that the application of an intermediate level of noise in the environment of a MEMS magnetic field sensor improves its detection capability of subthreshold signals via the stochastic resonance phenomenon.     

Detection of Pseudomonas aeruginosa using a wireless magnetoelastic sensing device

This paper describes the real-time quantification of Pseudomonas aeruginosa (P. aeru) concentrations using a wireless magnetoelastic sensing device. The sensor is fabricated by coating a magnetoelastic ribbon with a polyurethane protecting film. In response to an externally applied time varying magnetic field, the magnetoelastic sensor vibrates at a resonance frequency that can be remotely determined by monitoring the magnetic flux emitted by the sensor. The resonance frequency changes in response to properties changes of a liquid culture medium and bacteria adhesion to the sensor as P. aeru consumes nutrients from the culture medium in growth and reproduction. The effects of properties (conductivity, viscosity, mass) are investigated with quartz crystal microbalance (QCM), microscopy imaging, and conductivity measurement. Using the described technique we are able to directly quantify P. aeru concentrations of 10(3) to 10(8)cells/ml, with a detection limit of 10(3)cells/ml at a noise level of approximately 20 Hz. The lack of any physical connections between the sensor and the monitoring electronics facilitates aseptic operation, and makes the sensor platform ideally suited for monitoring bacteria from within, for example, sealed food containers.     

A T7exonuclease‐assisted target recycling amplification with graphene oxide acting as the signal amplifier for fluorescence polarization detection of human immunodeficiency virus (HIV) DNA

We report a fluorescence polarization (FP) platform for human immunodeficiency virus (HIV) DNA detection based on T7exonuclease‐assisted target recycling amplification with graphene oxide (GO) acting as a FP signal amplifier. In the sensing method, the presence of the target DNA leads to target recycling with the assistance of T7exonuclease, furthermore, the amplification products are absorbed onto the surface of GO, so the all FP values are enhanced by GO. More importantly, this FP sensor exhibits high detection sensitivity; under optimal conditions, the change in FP is linear with the concentration of the target DNA within a concentration range of 50–2000 pmol/L, and the detection limit of this method is as low as 38.6 pmol/L. This FP sensor also exhibits high selectivity, even single‐base mismatched DNA can be effectively discriminated from complementary target DNA. Above all, the proposed FP sensor may serve as a general platform for the sensitive assay of disease‐related genes. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Highly sensitive detection of organophosphorus insecticides using magnetic microbeads and genetically engineered acetylcholinesterase

This work presents a biosensor for organophosphorus pesticides based on immobilisation of a highly sensitive genetically engineered acetylcholinesterase (B394) by affinity interactions on metal chelate-functionalised magnetic microbeads. The developed sensor has been compared with those based on the widely used Electric eel cholinesterase and a classical entrapment procedure in a polyvinylalcohol-based matrix. The use of the B394 enzyme allowed lowering both IC50 and LOD by a factor of 100 when compared with Electric eel enzyme sensor. The oriented and site-specific immobilisation combined with the high specificity of the B349 mutant allows a more sensitive detection of insecticides, concentrations as low as 1.31(-11)M (IC10) being detected for both pesticides chlorpyriphos-oxon and chlorfenvinphos.     

Quantitative biomolecular sensing station based on magnetoresistive patterned arrays

The combination of magnetoresistive sensors and magnetic labeling of bioanalytes, which are selectively captured by their complementary antibody in the proximity of the sensor is a powerful method in order to attain truly quantitative immunological assays. In this paper we present a technical solution to exploit the existing spin valve technology to readout magnetic signals of bio-functionalized magnetic nanoparticles. The method is simple and reliable, and it is based on a discrete scan of lateral flow strips with a precise control of the contact force between sensor and sample. It is shown that the signal of the sensor is proportional to the local magnetization produced by the nanoparticles in a wide range of concentrations, and the sensitivity thresholds in both calibration samples and real immunorecognition assays of human chorionic gonadotropin hormone are well below the visual inspection limit (5.5 ng/ml). Furthermore the sample scanning approach and the reduced dimensions of the sensors provide unprecedented spatial resolution of the nanoparticle distribution across the supporting nitrocellulose strip, therefore enabling on-stick control references and multi-analyte capability.     

Platelet-derived growth factor oncoprotein detection using three-dimensional carbon microarrays

The potential of aptamers as ligand binding molecule has opened new avenues in the development of biosensors for cancer oncoproteins. In this paper, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor (PDGF-BB) oncoprotein detection is reported. The detection mechanism is based on the release of fluorophore (TOTO intercalating dye) from the target binding aptamer's stem structure when it captures PDGF. Amino-terminated three-dimensional carbon microarrays fabricated by pyrolyzing patterned photoresist were used as a detection platform. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5pmol. This detection strategy is promising in a wide range of applications in the detection of cancer biomarkers and other proteins.     

Glutamate detection from nerve cells using a planar electrodes array integrated in a microtiter plate

There is an increasing interest in new strategies for replacing animal tests in research. The use of cell cultures and integrated electrodes is seen as a promising alternative that could potentially solve this problem. In this work, we present a L-glutamate sensor based on a bienzyme redox hydrogel, capable of detecting the release of this excitatory neurotransmitter from adherently growing cells upon stimulation. The low working potential required for the operation of the sensor decreases the possibility of interference by easily oxidizable compounds always present in complex biological samples. A low detection limit of 0.5 microM L-glutamate, a response time of about 35 s, and a linear range of up to 60 microM are the main characteristics of the sensor. The system has been successfully employed to monitor the release of l-glutamate from HN10 and C6 cells upon stimulation with K(+)-ions. The developed integrated electrochemical platform will be used in future for drug screening and potentially for replacing animal models in neurological experiments.     

Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.

Covalent DNA-streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA-streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded DNA fragment or an enzyme and (ii) low-molecular weight biotin derivatives ("modulators") were coupled stepwise with the hybrid molecules. Modulators were D-biotin, aminobiotin, and biotin-fluorescein conjugate as well as a lysine-rich 10mer peptide, containing a biotin and a fluorescein substituent. These modulators were chosen to affected the hybridization properties of the DNA-streptavidin conjugates. As investigated by surface-plasmon resonance and microplate solid-phase hybridization measurements, D-biotin, biotin-fluorescein, and aminobiotin decreased the efficiency of hybridization with complementary, surface-bound oligonucleotides to a varying extent. The basic peptide increased the conjugate's hybridization efficiency. Moreover, it was demonstrated in two examples how modulators can be utilized as additional functional domains of streptavidin-based conjugates. First, fluorescein-containing modulators were used as hapten groups, allowing a sensitive detection by means of specific antibodies directed against the modulator. Second, the biotinylated peptide was used as a carrier molecule to attach multiple fluorogenic lanthanide-chelate groups to the streptavidin conjugate, enabling its sensitive detection by time-resolved fluorometry. The applicability of this kind of bioconjugation strategy to generate sensor-probes for gene detection assays was demonstrated.