Biomolecular Recognition Elements Research Articles

Microfabricated Magnetoelastic Biosensors for the Detection of Bacillus Anthracis Spores

Phage based magnetoelastic biosensors for wireless detection of Bacillus anthracis spores were fabricated. The biosensor is composed of a magnetoelastic resonator platform, and JRB7 phage, a genetically engineered bacteriophage that specifically binds with Bacillus anthracis spores, as the bio-molecular recognition element. The phage was engineered at Auburn University and immobilized onto all surfaces of the sensors. To achieve high sensitivity, micro-scale magnetoelastic particle sensor platforms were manufactured using microelectronics fabrication techniques. Real-time in liquid tests were conducted to determine the sensitivity and detection limit of the biosensors by exposing the biosensors to static solutions containing known concentrations of spores. Experimental results showed that the biosensor has a detection limit of approximately 100 spores per ml of liquid and sensitivity of 13 KHz per decade for the detection of Bacillus anthracis spores. Additionally, the phage based biosensors showed high binding affinity and specificity, and superior stability compared with antibody based biosensors.

Enzymatic Cleavage of Nucleic Acids on Gold Nanoparticles: A Generic Platform for Facile Colorimetric Biosensors

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.

A wireless biosensor using microfabricated phage-interfaced magnetoelastic particles

A micro-scale, free standing, wireless biosensor has been developed using magnetoelastic particles composed of an amorphous iron–boron binary alloy. Upon the application of an external magnetic field, these particles exhibit a characteristic resonance frequency, determined by their size and mass, due to the phenomena of magnetoelasticity. The particles are produced using the microelectronic fabrication techniques of photolithography and physical vapor deposition (sputtering). The biosensor is formed by coating the magnetoelastic particle with a thin layer of gold and immobilizing a biomolecular recognition element (bacteriophage) on the surfaces. Bacteriophage genetically engineered to bind Bacillus anthracis spores was used in this set of experiments as the detection probe. Once these targeted spores come into contact with the biosensor, the phage will bind selectively with only that pathogen, thereby increasing the particle's mass and causing a shift in the resonance frequency. Due to the magnetic nature of the sensing platform, this resonance frequency shift may be detected remotely by a wireless scanning device, presenting a distinct advantage over other techniques. A good correlation between the actual number of spores bound to the sensors and the calculated attached mass, based upon resonance frequency shifts, was obtained from the experiments.

Studies of the binding and signaling of surface-immobilized periplasmic glucose receptors on gold nanoparticles: A glucose biosensor application

Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur–gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV–Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 μM.

Dielectrophoretic Manipulation and Real‐Time Electrical Detection of Single‐Nanowire Bridges in Aqueous Saline Solutions

Dielectrophoretic manipulation of nanoscale materials is typically performed in nonionic, highly insulating solvents. However, biomolecular recognition processes, such as DNA hybridization and protein binding, typically operate in highly conducting, aqueous saline solutions. Here, we report investigations of the manipulation and real-time detection of individual nanowires bridging microelectrode gaps in saline solutions. Measurements of the electrode impedance versus frequency show a crossover in behavior at a critical frequency that is dependent on the ionic strength. We demonstrate that by operating above this critical frequency, it is possible to use dielectrophoresis to manipulate nanowires across electrode gaps in saline solutions. By using electrical ground planes and nulling schemes to reduce the background currents, we further demonstrate the ability to electrically detect bridging and unbridging events of individual nanowires in saline solutions. The ability to both manipulate and detect bridging events with electrical signals provides a pathway toward automated assembly of nanoscale devices that incorporate biomolecular recognition elements.

The effect of annealing and gold deposition on the performance of magnetoelastic biosensors

Magnetoelastic materials have a mass-sensitive, characteristic, resonant frequency that make them an ideal choice for application as the platform for a biosensor. The biosensor is formed by immobilizing a bio-molecular recognition element directly onto the surface of the magnetoelastic material. When exposed to the analyte, this bio-molecular recognition element binds the target species (spore or bacteria of interest), thereby increasing the mass and decreasing the resonance frequency of the sensor. This change in characteristic resonance frequency can be determined by monitoring remotely the magnetic flux in response to an applied, time varying, magnetic field with a pick-up coil. However, the environmental stability and bioactivity of the biosensor platform material are very important to performance of the overall biosensor. This research investigates the effects of annealing and deposition of a gold layer on the environmental stability and detection sensitivity of a magnetoelastic biosensor platform. The results show that deposition of a gold layer by sputtering additionally improved bioreactivity while maintaining a balance of environmental stability and detection sensitivity. Two annealing steps in a vacuum oven, one before and one after gold deposition, can effectively increase the magnetoelastic platform's environmental stability, as well as the Q-factor of the resonance signal.

Phage-Based Magnetoelastic Wireless Biosensors for Detecting Bacillus Anthracis Spores

A biosensor for the detection of biological warfare agents (Bacillus anthracis spores) was developed that combines the phage display technique with a magnetoelastic wireless detection platform. The affinity-based biosensor utilizes a phage-derived diagnostic probe as the biomolecular recognition element to capture target agents multivalently. Upon binding of the target agent to the sensor surface, the resonance frequency of the magnetoelastic biosensors decreases due to the additional mass of the target agent. Scanning electron microscopy was used to confirm binding of spores to the sensor surface. The sensitivity of the magnetoelastic acoustic sensor was tested to be 130 Hz per order of magnitude of spore concentration with a detection limit of 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> spores/ml. The specificity of the sensors was tested against spores of other closely related Bacillus species and a large preferential binding to Bacillus anthracis spores was observed. The longevity of the phage based biosensor was compared to traditional antibody based biosensors and found to exhibit a much longer life

A magnetoelastic resonance biosensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium

Mass-sensitive, magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless and remote. A biosensor for the detection of Salmonella typhimurium was constructed by immobilizing a polyclonal antibody (the bio-molecular recognition element) onto the surface of a magnetostrictive platform. The biosensor was then exposed to solutions containing S. typhimurium bacteria. Binding between the antibody and antigen (bacteria) occurred and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Sensors with different physical dimensions were exposed to different concentrations of S. typhimurium ranging from 10(2) to 10(9)CFU/ml. Detection limits of 5x10(3) CFU/ml, 10(5) CFU/ml and 10(7) CFU/ml were obtained for sensors with the size of 2 mmx0.4 mmx15 microm, 5 mmx1 mmx15 microm and 25 mmx5 mmx15 microm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.

Microfabricated, Wireless, Magnetoelastic Micro-Particles for the Detection of Bacillus Anthracis Spores

ABSTRACTMagnetoelastic resonance biosensors were fabricated by immobilizing a bio-molecular recognition element onto the surface of Fe79B21 magnetoelastic particles (MEP). These sensors can be measured wirelessly and remotely for both in-air and in-liquid bacteria detection. Filamentous bacterio-phage that was selected specifically for the detection of Bacillus anthracis spores was employed as the biomolecular recognition element and immobilized onto the MEPs' surfaces. Attachment of the spores to the sensor surface due to specific phage-spore binding results in a shift in the resonance frequency of the biosensor. Insitu measurement of the resonance frequency of biosensors of 5×100×500 microns were used to determine the sensor response as a function of spore concentrations of 103 to 108 cfu/ml. Specificity of the sensor was evaluated by conducting tests using a mixture of Bacillus anthracis Sterne strain, Bacillus cereus and Bacillus megaterium spores.

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.

Present and future of surface plasmon resonance biosensors.

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves-surface plasmon-polaritons-to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.

Surface plasmon resonance biosensor based on integrated optical waveguide

We report a sensor based on spectral interrogation of surface plasmon resonance (SPR) in an integrated optical waveguide-coupled SPR sensing device. We present theoretical analysis of the integrated optical SPR sensor structure leading to a device design optimized for operation in aqueous environments. We demonstrate that the fabricated laboratory prototype of the sensor is capable of measuring bulk refractive index changes smaller than 1.2×10 −6. In conjunction with specific biomolecular recognition elements (monoclonal antibodies against human chorigonadotropin (hCG)) the sensor is used for the detection of hCG. The sensor is demonstrated to be capable of detecting 2 ng of hCG present in 1 ml of 1% bovine serum albumin solution.

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces.

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected omega-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.