Multiplex Immunoassays Utilizing Differential Affinity Using Aptamers Generated by MARAS.

Disease diagnosis typically requires to determine concentration of multiple biomarkers in patient serums. Here, a novel method for multiplex immunoassays is proposed and the feasibility is demonstrated. The method utilizes the differential affinity between aptamers and multiple analytes for multiplex immunoassays. During the selection, aptamers capable of binding to multiple analytes with different affinities are screened from a random oligonucleotide library using the MARAS procedure with different magnetic field conditions for different target analytes. During the detection, the same magnetic field conditions are applied to differentiate different target analytes in blind serums. The results show that the recovery rates of the spiked targets in BD buffer and blind serums are similar. Moreover, there is a minimal interference resulting from non-specific binding of molecules in serums other than the target molecules. Therefore, the use of differential affinities between aptamers and different analytes for multiplex immunoassays is proved to be feasible.

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection.

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5' and 3' termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Magnetic-assisted rapid aptamer selection (MARAS) for generating high-affinity DNA aptamer using rotating magnetic fields.

A new SELEX protocol for the development of DNA aptamers has been demonstrated, referred to as magnetic-assisted rapid aptamer selection (MARAS). This method uses magnetic beads and an externally applied rotating magnetic field to provide the competitive mechanism for the selection aptamers with different affinities to the molecular target. The MARAS protocol efficiently generated aptamers with high affinity and specificity for C-reactive protein, a common cardiovascular disease indicator. The binding affinities of the selected aptamers could be varied by changing the frequency of the externally applied rotating magnetic field and optimal cases bound with low-nanomolar dissociation constants.

A novel protocol for generating high-affinity ssDNA aptamers by using alternating magnetic fields.

BACKGROUND: Aptamers that have been generated using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) process have been found to have high specificity and affinity with molecular targets, and, as a result, have found use in diagnostic and therapeutic applications. The SELEX protocol generally requires 5 to 15 rounds of selection to generate high-affinity aptamers; however, the standard protocol is labor-intensive and time-consuming. Here, we propose a method for DNA aptamer screening, in which the evolutionary process is completely abandoned. METHODS: The working principle of this new protocol, Magnetic-Assisted Rapid Aptamer Selection (MARAS), is discussed. We used C-reactive protein, a common cardiovascular disease (CVD) indicator, as a molecular target. An externally applied alternating magnetic field, aided by target-bound magnetic nanoparticles, was used to provide the competitive mechanism for the selection of DNA aptamers with different affinities to the target protein. RESULTS: The range of the dissociation constants of the screened aptamers was approximately several nMs, depending on the frequency and the field strength of the externally applied alternating magnetic field. We also compared the diagnostic applicability of the aptamers generated by the proposed MARAS protocol with an enzyme-linked immunosorbent assay (ELISA), using antibodies. CONCLUSIONS: The proposed MARAS protocol efficiently generates aptamers that have high affinity and specificity with molecular targets. In addition, the proposed protocol represents significant time savings, as it can be completed in less than an hour. Furthermore, due to the simplicity of the MARAS protocol, we suggest that the proposed process could be easily automated.

Combined plasma biomarkers for diagnosing mild cognition impairment and Alzheimer's disease.

A highly sensitive immunoassay, the immunomagnetic reduction, is used to measure several biomarkers for plasma that is related to Alzheimer's disease (AD). These biomarkers include Aß-40, Aß-42, and tau proteins. The samples are composed of four groups: healthy controls (n=66), mild cognitive impairment (MCI, n=22), very mild dementia (n=23), and mild-to-serve dementia, all due to AD (n=22). It is found that the concentrations of both Aß-42 and tau protein for the healthy controls are significantly lower than those of all of the other groups. The sensitivity and the specificity of plasma Aß-42 and tau protein in differentiating MCI from AD are all around 0.9 (0.88-0.97). However, neither plasma Aß-42 nor tau-protein concentration is an adequate parameter to distinguish MCI from AD. A parameter is proposed, which is the product of plasma Aß-42 and tau-protein levels, to differentiate MCI from AD. The sensitivity and specificity are found to be 0.80 and 0.82, respectively. It is concluded that the use of combined plasma biomarkers not only allows the differentiation of the healthy controls and patients with AD in both the prodromal phase and the dementia phase, but it also allows AD in the prodromal phase to be distinguished from that in the dementia phase.

Anti-CEA-functionalized superparamagnetic iron oxide nanoparticles for examining colorectal tumors in vivo.

Although the biomarker carcinoembryonic antigen (CEA) is expressed in colorectal tumors, the utility of an anti-CEA-functionalized image medium is powerful for in vivo positioning of colorectal tumors. With a risk of superparamagnetic iron oxide nanoparticles (SPIONPs) that is lower for animals than other material carriers, anti-CEA-functionalized SPIONPs were synthesized in this study for labeling colorectal tumors by conducting different preoperatively and intraoperatively in vivo examinations. In magnetic resonance imaging (MRI), the image variation of colorectal tumors reached the maximum at approximately 24 h. However, because MRI requires a nonmetal environment, it was limited to preoperative imaging. With the potentiality of in vivo screening and intraoperative positioning during surgery, the scanning superconducting-quantum-interference-device biosusceptometry (SSB) was adopted, showing the favorable agreement of time-varied intensity with MRI. Furthermore, biological methodologies of different tissue staining methods and inductively coupled plasma (ICP) yielded consistent results, proving that the obtained in vivo results occurred because of targeted anti-CEA SPIONPs. This indicates that developed anti-CEA SPIONPs owe the utilities as an image medium of these in vivo methodologies.

In vivo screening of hepatocellular carcinoma using AC susceptibility of anti-alpha fetoprotein-activated magnetic nanoparticles.

With antibody-mediated magnetic nanoparticles (MNPs) applied in cancer examinations, patients must pay at least twice for MNP reagents in immunomagnetic reduction (IMR) of in vitro screening and magnetic resonance imaging (MRI) of in vivo tests. This is because the high maintenance costs and complex analysis of MRI have limited the possibility of in vivo screening. Therefore, this study proposes novel methods for in vivo screening of tumors by examining the AC susceptibility of bound MNPs using scanning superconducting-quantum-interference-device (SQUID) biosusceptometry (SSB), thereby demonstrating high portability and improved economy. The favorable agreement between in vivo tests using SSB and MRI demonstrated the feasibility of in vivo screening using SSB for hepatocellular carcinoma (HCC) targeted by anti-alpha fetoprotein (AFP)-mediated MNPs. The magnetic labeling was also proved by in vitro tests using SSB and biopsy assays. Therefore, patients receiving bioprobe-mediated MNPs only once can undergo in vivo screening using SSB in the future.

Use of immunomagnetic reduction for C-reactive protein assay in clinical samples.

BACKGROUND: Magnetic nanoparticles biofunctionalized with antibodies are able to recognize and bind to the corresponding antigens. In this work, anti-C-reactive protein (CRP) antibody was covalently conjugated onto the surface of magnetic nanoparticles to label CRP specifically in serum. METHODS: The level of serum CRP was detected by immunomagnetic reduction (IMR) assay, which identifies the changes in the magnetic signal representing the level of interaction between antibody-conjugated magnetic nanoparticles and CRP proteins. To investigate the feasibility of IMR for clinical application, pure CRP solutions and 40 human serum samples were tested for IMR detection of CRP to characterize sensitivity, specificity, and interference. RESULTS: In comparison with the immunoturbidimetry assay, the results of the IMR assay indicated higher sensitivity and had a high correlation with those of the current immunoturbidimetry assay. CONCLUSION: We have developed a novel and promising way to assay CRP in human serum using immunomagnetic reduction in clinical diagnosis.

Characteristics of magnetic labeling on liver tumors with anti-alpha-fetoprotein-mediated Fe3O4 magnetic nanoparticles.

For preoperative and intraoperative detection of tumor distribution, numerous multimodal contrast agents, such as magnetic nanoparticles (MNPs) with several examination indicators, are currently in development. However, complex materials, configuration, and cost are required for multimodal contrast agents, accompanied by a high possibility of toxicity and low popularity in clinics. Nevertheless, the magnetic labeling of MNPs using bioprobes should be feasible not only in preoperative magnetic resonance imaging (MRI), but also in intraoperative examination based on other magnetic properties. In this study, anti-alpha-fetoprotein (AFP)-mediated Fe(3)O(4) MNPs, injected into mice with liver tumors, were used to examine the characteristics of magnetic labeling. Using MRI and scanning superconducting-quantum-interference-device biosusceptometry (SSB), based on alternating current (AC) susceptibility, the magnetic labeling occurred significantly on the first day post-injection of anti-AFP magnetic fluid (MF), and then decreased over time. However, for both MF without antibodies and an anti-carcinoembryonic antigen MF, no magnetic labeling occured on the first day of their respective post-injection. The favorable agreement indicates that magnetic labeling possesses two magnetic characteristics: distortion of the imaging field and AC susceptibility. In addition, the results of the biopsy tests, anti-AFP staining, and Prussian blue staining show the same dynamics as those of magnetic methodologies and prove that bound MNPs on tumor tissue are rotatable by an AC magnetic field to express AC susceptibility. Therefore, with the simple configuration of antibody-mediated MNPs, magnetic labeling is also feasible for intraoperative examinations using SSB with high mobility and sensitivity.

Feasibility studies for assaying alpha-fetoprotein using antibody-activated magnetic nanoparticles.

Some previous reports have already shown the characterizations of immunomagnetic reduction (IMR). The assay technology involves the utilities of biofunctionalized magnetic nanoparticles to label target biomolecules. However, the detection threshold and interference tests for IMR have not been investigated in detail. In this study, alpha-fetoprotein (AFP) was used as a target biomolecule. The signals for AFP solutions of various concentrations, or with interfering materials, were detected via IMR. These samples were also used for characterizing the detection threshold and interference with enzyme-linked immunosorbent assay (ELISA). The results of assaying AFP level with IMR and ELISA were compared. The detection threshold for assaying AFP with IMR was found to be 3 ng/mL, which is 15 times lower than that of ELISA, and definitely suppresses false negative. For the interfering materials noted commonly in serum such as hemoglobin, bilirubin, triglyceride, and vascular endothelial growth factor, there was no detectable interfering effect when assaying AFP with IMR. Several serum samples from normal people and liver-tumor-bearing patients were used for the detections of AFP concentration via IMR. These results reveal the feasibilities of assaying AFP in blood using IMR, as well as achieving high-sensitive and high-specific assay for AFP.

Exploration of the relationship between the tumor burden and the concentration of vascular endothelial growth factor in liver-cancer-bearing animals using immunomagnetic reduction assay.

To achieve early-stage diagnosis, a high-sensitivity assay method is needed. As a biomarker, vascular endothelial growth factor (VEGF) has played a growing role in diagnosing and treating hepatocellular carcinoma (HCC). In this work, an immunomagnetic reduction (IMR) through bio-functionalized magnetic nanoparticles and a high-temperature superconducting-quantum-interference-device magnetometer were utilized for quantitative detection of low-concentration VEGF in serum from rats with HCC. The precision and accuracy of IMR on VEGF were characterized. Further, the results of assaying VEGF in the serum of rats were compared with those of using enzyme-linked immunosorbant assay (ELISA). It was found the correlations between the detected VEGF concentration in the rat serum and tumor burdens were 0.99 and 0.90 for IMR and ELISA, respectively, within the range from 2 pg/ml to 8000 pg/ml of VEGF concentration.

Biofunctionalized magnetic nanoparticles for specifically detecting biomarkers of Alzheimer's disease in vitro.

Magnetic nanoparticles biofunctionalized with antibodies against ß-amyloid-40 (Aß-40) and Aß-42, which are promising biomarkers related to Alzheimer's disease (AD), were synthesized. We characterized the size distribution, saturated magnetizations, and stability of the magnetic nanoparticles conjugated with anti-Aß antibody. In combination with immunomagnetic reduction technology, it is demonstrated such biofunctionalized magnetic nanoparticles are able to label Aßs specifically. The ultralow-detection limits of assaying Aßs in vitro using the magnetic nanoparticles via immunomagnetic reduction are determined to a concentration of ∼10 ppt (10 pg/mL). Further, immunomagnetic reduction signals of Aß-40 and Aß-42 in human plasma from normal samples and AD patients were analyzed, and the results showed a significant difference between these two groups. These results show the feasibility of using magnetic nanoparticles with Aßs as reagents for assaying low-concentration Aßs through immunomagnetic reduction, and also provide a promising new method for early diagnosis of Alzheimer's disease from human blood plasma.

Magnetically enhanced high-specificity virus detection using bio-activated magnetic nanoparticles with antibodies as labeling markers.

This study describes magnetically driven suppression of cross-reactions among molecules. First, the magnetic nanoparticles are coated with bio-probes and dispersed in liquid. The bio-probes can then bind with homologous or heterologous bio-targets. When alternating-current (ac) magnetic fields are applied, magnetic nanoparticles rotate driven by ac magnetic fields. Thus, the bio-targets bound on the surface of magnetic nanoparticles experience a centrifugal force. The centrifugal force can be manipulated by adjusting the angular frequency of the rotating magnetic nanoparticles. The angular frequency is determined by the applied ac magnetic field frequency. Since the binding force for good binding is much higher than that of poor binding, frequency manipulation is needed for the centrifugal force to be higher than the poor-binding force but lower than the good-binding force. Therefore, poor binding which contributes to cross reactions between molecules can be suppressed efficiently by control of the ac magnetic field frequency.

Simultaneous identification of the low-field-induced tiny variation of complex refractive index for anisotropic and opaque magnetic-fluid thin film by a stable heterodyne Mach-Zehnder interferometer.

We use a heterodyne Mach-Zehnder interferometer to simultaneously and simply measure the complex refractive index by only normal incidence on the specimen, instead of using a complicated measurement procedure or instrument that only measures the real or imaginary part of the complex refractive index. To study the tiny variation of the complex refractive index, the small complex refractive-index variation of a rare-concentration magnetic-fluid thin film, due to a weak field of less than 200 Oe, was processed by this interferometer. We also present the wavelength trend of the complex refractive index of magnetic fluids to verify the appearance of the slight change in a small wavelength range.

Ultra-highly sensitive and wash-free bio-detection of H5N1 virus by immunomagnetic reduction assays.

A platform for assaying avian influenza H5N1 viruses that involves measuring the ac immunomagnetic reduction of a magnetic reagent mixed with a detected sample is developed. The magnetic reagent contained magnetic nanoparticles coated with antibodies. To achieve an ultra-high sensitivity assay, a system utilizing a high-transition-temperature superconducting quantum interference device was used to sense the immunomagnetic reduction of the reagents. The results confirmed the ultra-high sensitivity of the immunomagnetic reduction assay on H5N1.

Wash-free, antibody-assisted magnetoreduction assays of orchid viruses.

This study demonstrates the feasibility of wash-free magnetoreduction assays (MRA) of orchid viruses. A magnetic reagent, consisting of magnetic beads coated with antibodies and dispersed in water, was synthesized. By using a mixed-frequency alternative-current (ac) magnetosusceptometer, differences in the magnetic susceptibilities of the magnetic reagent before and after the addition of orchid solutions were measured. The results show significant advantages for MRA of orchid viruses.

Ex vivo magnetofection with magnetic nanoparticles: a novel platform for nonviral tissue engineering.

Several methods have been described to introduce DNA expression vectors into mammalian cells both in vitro and in vivo. Each system has benefits and limitations, and to date there is still no ideal method for gene transfer. In this study, we introduced a novel method of gene transfer by using Fe3O4 nanoparticles. The magnetic nanoparticles composed of Fe3O4, and the transfected genes used are Lac Z and enhanced green fluorescence protein gene (EGFG). Four different groups of preparations included in this study were homemade liposome-enveloped EGFP-DNA/Fe3O4, homemade liposome EGFP-DNA gene without magnetic Fe3O4 nanoparticles, lipofectamine 2000-enveloped EGFP-DNA, and EGFP-DNA gene only. Mice osteoblast and He99 lung cancer cell line were used as host cells for gene transfection. The time-dependent EGFP gene expression was monitored and analyzed. The results showed that the diameter of the complex was less than 100 nm. There was no cytotoxicity observed at any of the magnetic Fe3O4 nanoparticle concentrations tested. In the presence of magnetic field, the liposome-enveloped EGFP-DNA/Fe3O4 complex exhibited a much higher efficiency for transfecting EGFP-DNA into osteoblast cells under external magnetic fields. The gene can be transfected into cells with an aid of magnetic vectors and magnetic force. Under a gradient magnetic field, the efficiency of magnetofection is enhanced as compared to that without magnetic field.

Focusing concave lens using photonic crystals with magnetic materials.

The guided modes lying in the upper gap-edge band in the photonic band structure of photonic crystals have negative values of refractive index. This feature generates many interesting optical phenomena, and some spectacular photonic devices such as focusing slabs have been developed. We report the design of a photonic-crystal, planoconcave lens for focusing incident parallel light, and theoretically analyze the chromatic aberrations for TM and TE modes. In addition to dielectric photonic crystals, the chromatic aberration of a magnetic photonic-crystal planoconcave lens was investigated because the magnetic permeability may also contribute to the periodic index contrast in photonic crystals, especially at long wavelengths. A significant difference was found in the chromatic aberration for a TM mode propagating in a dielectric than in a magnetic photonic-crystal planoconcave lens.

Designing optical-fiber modulators by using magnetic fluids.

To reduce interface loss between optical fibers and devices in telecommunication systems, the development of an optical-fiber-based device that can be fused directly with fibers is important. A novel optical modulator consisting of a bare fiber core surrounded by magnetic fluids instead of by a SiO2 cladding layer is proposed. Applying a magnetic field raises the refractive index of the magnetic fluid. Thus we can control the occurrence of total reflection at the interface between the fiber core and the magnetic fluid when light propagates along the fiber. As a result, the intensity of the outgoing light is modulated by variation in field strength. Details of the design, fabrication, and working properties of such a modulator are presented.

Tunable diffraction of magnetic fluid films and its potential application in coarse wavelength-division multiplexing.

When an external magnetic field is applied parallel to the film surface of a magnetic fluid film, a high-quality one-dimensional periodic chain structure is formed when the field strength reaches a certain level. With a periodic chain structure in the magnetic fluid film, an incident light is diffracted onto the magnetic thin film. The results show that the one-dimensional periodic chain structure in the magnetic fluid film can serve as an optical grating. Further investigations reveal the feasibility of developing tunable coarse wavelength-division multiplexing by utilizing a periodic chain structure.