Detection of C-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles

We report a detection method for C-reactive protein (CRP) based on competitive immunoassay using magnetic nanoparticles under magnetic fields. Functional magnetic nanoparticles were prepared and conjugated with anti-CRP for immunoassay. Magnetic nanoparticles labeled with anti-CRP were flowed through a separation channel to form depositions for selective capture of CRP under magnetic fields. Free CRP and a fixed number of CRP-labeled particles were used to compete for a limited number of anti-CRP binding sites on the magnetic nanoparticles. The deposited percentages of CRP-labeled particles at various concentrations of free CRP were determined and used as a reference plot. The determination of CRP in the unknown sample was deduced from the reference plot using the deposited percentages. The running time was less than 10 min. The CRP concentration of serum sample was linearly over the range of 1.2-310 microg/mL for deposited percentages of CRP-labeled particles. The detection limit of this method was 0.12 microg/mL which was approximately 8-fold lower than the typical clinical cutoff concentration (1 microug/mL). This method can provide a fast, simple, and sensitive way for protein detection based on competitive immunoassay using magnetic nanoparticles under magnetic fields.     

Coupling stimuli-responsive magnetic nanoparticles with antibody-antigen detection in immunoassays

Because current homogeneous immunoassays show some limitations, particularly low sensitivity, we developed a new immunoassay to overcome these limitations. The approach was based on magnetic nanoparticles with a thermoresponsive polymer layer, a negatively charged polymer, and streptavidin-biotin-based antibody-antigen detection and yielded higher sensitivity than commonly used heterogeneous immunoassays. Because no special equipment is needed, it can be applied to currently available absorbance-based systems for high-throughput assays.     

Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles

Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria are described for the first time by using magnetic micro- and nanoparticles. The covalent immobilization of a commercial monoclonal antibody toward the HRP2 protein in magnetic beads and nanoparticles was evaluated and compared. The immunological reaction for the protein HRP2 was successfully performed in a sandwich assay on magnetic micro- and nanoparticles by using a second monoclonal antibody labeled with the enzyme, horseradish peroxidase (HRP). Then, the modified magnetic particles were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with the electrochemical magneto immunosensors was successfully evaluated and compared with a novel magneto-ELISA based on optical detection using spiked serum samples. Improved sensitivity was obtained when using 300 nm magnetic nanoparticles in both cases. The electrochemical magneto immunosensor coupled with magnetic nanoparticles have shown better analytical performance in terms of limit of detection (0.36 ng mL(-1)), which is much lower than the LOD reported by other methods. Moreover, at a low level of HRP2 concentration of 31.0 ng mL(-1), a signal of 15.30 μA was reached with a cutoff value of 0.34 μA, giving a clear positive result with a non-specific adsorption ratio of 51. Due to the high sensitivity, this novel strategy offers great promise for rapid, simple, cost-effective, and on-site detection of falciparum malaria disease in patients, but also to screen out at-risk blood samples for prevention of transfusion-transmitted malaria.     

Fluorogenic nanocrystals for highly sensitive detection of C-reactive protein

A solid-phase sandwich fluorescence immunoassay using nanocrystals of a fluorogenic precursor, fluorescein diacetate (FDA), conjugated with monoclonal antibodies for the detection of C-reactive protein (CRP), is described. FDA nanocrystals were coated with distearoylglycerophosphoethanolamine (DSPE), modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. CRP was chosen as a model analyte because of its widely accepted role as a marker for acute inflammation and prospective heart failure. A low limit of detection (1.10 microg l(-1)) and high precision (CV = 2.72-9.48%) were achieved. Following the immunoreaction, the monoclonal anti-CRP conjugated nanocrystals were released by hydrolysis and dissolution instigated by the addition of a large volume of organic solvent-sodium hydroxide mixture. Using human serum samples from 66 patients with high heart attack risk and 19 healthy blood donors, this CRP fluorescence immunoassay showed a good correlation to the commercially available, turbidimetric immunoassay for CRP. This result was corroborated by the Bland-Altman plot that showed a mean difference between the two methods of only 0.36+/-1.46 mg l(-1). The study demonstrates that the organic fluorogenic FDA nanocrystals can be applied for the detection of CRP, which is a clinically interesting plasma protein with a low limit of detection.     

In-situ detection of C-reactive protein using silicon nanowire field effect transistor

Label-free, sensitive, and real-time c-reactive protein (CRP) sensor was fabricated using p-type silicon nanowire (SiNW) based structures configured as field effect transistors (FET) using the conventional 'top-down' semiconductor processes. The width of SiNWs were distributed 80 nm to 400 nm. Among them to improve signal-to-noise ratio and sensitivity of SiNW FET, 221 nm-SiNW was chosen for biosensing of CRP. Antibody of c-reactive protein (anti-CRP) was immobilized on the SiNW surface through polydimethylsiloxane (PDMS) microfluidic channel for detection of CRP. Specific binding of CRP with anti-CRP on the SiNW surface caused a conductance change of SiNW FET and various injections from 10 and 1 microg/ml to 100 ng/ml solutions of CRP resulted in the conductance changes from 39 and 25 to 16%, respectively. Label-free, in-situ and very sensitive electrical detection of CRP was demonstrated with the prepared SiNW FET.     

基于磁导率方法的钢板裂纹检测

为探究磁导率检测技术对宏观裂纹缺陷的检测能力,设计磁导率检测平台,建立磁导率检测裂纹的物理模型。以刻有人工裂纹的45~#钢板为试验对象,研究不同的激励电压对检测信号的影响,分析传感器不同检测方向的信号特征,从正反两面对钢板裂纹进行试验研究。试验表明:磁导率检测方法完成对钢板表面和背面裂纹的检测;检测钢板背面时,该方法可以检测2.5 mm表面厚度下的裂纹,具有很高的信噪比。该研究成果可拓展磁导率检测技术的应用领域,为钢板裂纹的正背面检测提供一种新的方法。     

Strategy and its implications of protein bioanalysis utilizing high-resolution mass spectrometric detection of intact protein

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.     

Evaluation of 2-methacryloyloxyethyl phosphorylcholine polymeric nanoparticle for immunoassay of C-reactive protein detection

To prepare novel 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymeric nanoparticle (MPC-PNP), water-soluble amphiphilic phospholipid polymer, poly [MPC-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP) (PMBN)], which has active ester groups for bioconjugation on the side chains, was synthesized. MPC-PNP was prepared by a solvent evaporation technique where the poly(l-lactic acid) was used as core and PMBN was applied as an emulsifier and a surface modifier under systematical design of well-arranged phospholipids polar groups in its surface. Characteristics for MPC-PNP were thoroughly investigated with dynamic light scattering, electrophoresis light scattering, X-ray photoelectron spectroscopy, and field emission scanning electron microscopy measurements. Through a protein adsorption test, the phosphorylcholine group on the surface of MPC-PNPs, which had their active ester groups substituted by glycine, were shown to suppress the nonspecific adsorption of bovine serum albumin. These particles were used for C-reactive protein (CRP) detection, where anti-CRP monoclonal antibodies were immobilized on the MPC-PNP using the active ester group, while the remaining active ester groups were thoroughly reacted with glycine. The detection limit about serum-free CRP in the calibration curve was shown to extend from 0.01 to 10 mg/dL when anti-CRP antibody immobilized MPC-PNP was used for serum-free CRP detection. This compares favorably with measurement using polystyrene nanoparticles that were shown to detect from 0.1 to 10 mg/dL by an immunoagglutination technique. Also, for the detection of CRP in serum, MPC-PNP was shown to give the same calibration curve explained by the efficient suppression of nonspecific binding. Furthermore, denaturation of immobilizing anti-CRP antibody on the MPC-PNP hardly occurred despite increasing the temperature. It is concluded that MPC-PNP is unique due to the design of its interfacial properties, also it will perform well in a diagnostic immunoassay because of its optimized material properties.     

CdSe and CdSe/ZnS quantum dots for the detection of C-reactive protein

In this study, a method for the detection of C-reactive protein (CRP) using CdSe and CdSe/ZnS quantum dots (QDs) is proposed. CdSe and CdSe/ZnS core-shell QDs are synthesised by using 2-mercaptosuccinic acid (MSA) as a capping agent. These QDs were then subjected to various characterisation studies, namely X-ray diffraction and transmission electron microscope for size and structure, Fourier transform infrared spectroscopy for the confirmation of functional groups, ultraviolet–visible absorption and fluorescence spectroscopy for optical characteristics and dynamic light scattering for hydrodynamic changes of QDs. Two biochemical mixtures were developed: one by mixing blood serum containing CRP and CdSe-phosphorylethanolamine (PEA) and the other by mixing blood serum with CdSe/ZnS-PEA. When these mixtures are observed for fluorescence due to interaction of QDs with CRP, a correlation between changes in fluorescence for different concentrations of CRP is noted. The result demonstrates that CRP can be detected with the help of QDs without using any antibodies.     

Sensitive affimer and antibody based impedimetric label-free assays for C-reactive protein

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. Levels greater than 3 μg/mL serum have hitherto been considered to indicate pathology, but there is increasing interest in assessments between 0.1 and 10 μg/mL, which have been found to correlate with severity of risk for cardiovascular disease. We report herein the generation of both antibody and Affimer based impedance immunoassays for CRP that are substantially more sensitive than clinically utilized immunonephelometry and immunoturbidity assessments. Significant in this study is not only the use of a constrained peptide to detect a clinically important target but also that derived electrochemical impedance assays can be highly sensitive even with probes whose relatively weak (μM) affinities are not amenable to target detection by surface plasmon resonance (SPR). Key to this finding is acknowledging that receptive surfaces of comparatively low initial steric bulk and charge transfer resistance are especially primed to be highly responsive to target binding in electroanalytical assays of this type.     

基于磁记忆弱漏磁效应的钢绞线腐蚀检测

为明确缺陷对试件表面磁场的影响,该文对30Cr合金钢进行了逐级加载拉伸试验,在试件表面提前人为预制了缺陷,利用TSC-1M-4型磁检测仪采集磁场信号,研究了试件表面磁记忆信号在缺陷处的特征表现。切向磁记忆信号对试件的局部屈服更敏感。对其进行分析的结果表明：试件表面的磁记忆信号在缺陷边界处有明显的变化,通过表面磁场分布可以对试件的应力集中状况进行评价。     

基于低场磁共振技术的糖溶液检测

采用低场核磁共振分析测试技术研究糖溶液的自旋-自旋弛豫特性.利用Carr-Purcell-MeiboomGill(CPMG)序列采集糖溶液的核磁共振回波信号,以多重指数衰减模型对采集的信号数据进行分析,探讨糖溶液中含糖量的多少与自旋-自旋弛豫时间的相关性,采用加权几何平均法计算不同浓度糖溶液的自旋-自旋弛豫时间.实验结果表明,蔗糖、葡萄糖和果糖的亲水性均能改变蒸馏水的自旋-自旋弛豫时间,并且,随着含糖量的升高,自旋-自旋弛豫时间呈指数降低;蔗糖、葡萄糖和果糖的相关系数均达到0.9以上.     

Detection of lysozyme magnetic relaxation switches based on aptamer-functionalized superparamagnetic nanoparticles

Magnetic relaxation switch (MRSw) detection is based on aggregate formation or dissociation when magnetic nanoparticles (MNPs) bind to target molecules. In the aggregated state, the dephasing rate of nearby proton spins is higher than in the dispersed state, resulting in a decrease in the spin-spin relaxation time, T(2). In this work, an MRSw-based nanosensor for lysozyme (Lys) protein detection was achieved using iron oxide nanoparticles conjugated with either Lys aptamer or linker DNA, which can hybridize with the extended part of the aptamer to form clusters. Upon the addition of Lys, the aptamers bind with their targets, leading to disassembly of clusters and an increase in T(2). A detection limit in the nanomolar range was achieved for Lys detection in both buffer and human serum. The determination of Lys level in different types of cancer cell lysates was also performed to demonstrate detection in real clinical samples.     

Impedimetric biosensor based on magnetic nanoparticles for electrochemical detection of dopamine

One of the difficulties which limit the use of electrochemical sensors for detection of dopamine is the interference from ascorbic acid. We have sought to address this problem through the synthesis and characterization of a suitable electrode material based on magnetic nanoparticles. The interference from the ascorbic acid was overcome by fabricating a negatively charged electrode surface using PEGylated arginine functionalized magnetic nanoparticles (PA-MNPs). The nanoparticles were characterized by various techniques viz., X-ray diffraction, FT-Infrared spectroscopy, transmission electron microscopy and vibrating sample magnetometer. The electrochemical behavior of the proposed sensor was investigated by cyclic voltammetry and the sensor showed high sensitivity and selectivity for dopamine. The response mechanism of the modified electrode is based on the interaction between the negatively charged electrode and the positively charged dopamine. Under optimized conditions, linear calibration plots were obtained for amperometric detection of dopamine (DA) over the concentration range of 1–9 mM dopamine, with a linear correlation coefficient of 0.9836, sensitivity of 121 μA/mM and a detection limit of 7.25 μM. Electrochemical impedance spectroscopy (EIS) has been used to study the interface properties of modified electrodes. The value of the polarization resistance (R_p) increases linearly with dopamine concentration in the range of 10 μM to 1 mM and the limit of detection (LOD) was calculated to be 14.1 μM. High sensitivity and selectivity, micromolar detection limit, high reproducibility, along with ease of preparation of the electrode surface make this system suitable for the determination of DA in pharmaceutical and clinical preparations.     

基于弱磁技术的火车轮踏面裂纹检测

针对火车轮踏面易萌生细小裂纹的问题,提出一种无需励磁,利用地磁场磁化的无损检测方法.根据火车轮踏面结构设计探头工装,利用弱磁检测仪器采集车轮踏面磁感应强度信号,分析预制裂纹缺陷的磁异常信号特征,对比不同深度裂纹的磁异常信号幅值,通过傅里叶最小二乘拟合得到缺陷深度的定量算法,根据极值差法与拉依达准则对火车轮踏面进行智能识...     

Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays

We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 microL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 microg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 microL of sample volume was used, a detection limit of 0.001 microg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.     

Detection of beta-amyloid (1-42) on protein array based on electrical detection technique using scanning tunneling microscopy

In this study an immuno-array for Abeta42 based on scanning tunneling microscopy (STM) was developed using conjugated gold nanoparticle (Au-NP) and antibody (Ab) complex. Fragmented monoclonal Ab against Abeta42 was allowed to immobilize on the Au-dot arrays followed by its target protein Abeta42 and Au NP and Ab complex. The surface structure of Au-NP and Ab complex on Au-dots was investigated with Atomic Force Microscopy and the current profile of fabricated immunosensing element was investigated with STM. The power spectrum derived from the current profile was found to be increasing with higher concentrations of Abeta42 having a detection limit of 100 fg/ml. The proposed technique can be a promising method to construct the highly sensitive and efficient protein chip of immunosensors arrays.     

Competitive immunoassays for simultaneous detection of metabolites and proteins using micromosaic patterning

New high-throughput immunoassay methods for rapid point-of-care diagnostic applications represent an unmet need and current focus of numerous innovative methods. We report a new micromosaic competitive immunoassay developed for the analysis of the thyroid hormone thyroxine (T4), inflammation biomarker C-reactive protein (CRP), and the oxidative damage marker 3-nitrotyrosine (BSA-3NT) on a silicon nitride substrate. To demonstrate the versatility of the method, both direct and indirect format competitive immunoassays were developed and could be applied simultaneously for single samples. Signals from standard solutions were fit to a logistic equation, allowing simultaneous detection of T4 (7.7-257.2 nM), CRP (0.3-4.2 microg/mL), and BSA-3NT (0.03-22.3 microg/mL). Total assay time including sample introduction, washing, and fluorescence measurement was less than 45 min. Dissociation constants for affinity pairs in the system have been estimated using regression. This proof-of-concept experiment shows that both small and macromolecular biomarkers can be quantified from a single sample using the method and suggests that groups of clinically related analytes may be analyzed by competitive micromosaic immunoassay techniques.     

基于电/磁转换的海洋电场检测方法

传统的直接电测量方法受1/f噪声限制,在低频处信号与噪声发生混叠,难以实现高水平低噪声测量。该文提出一种基于电/磁转换的海洋电场低噪声检测方法,将电信号转化为磁信号,通过微机械的方法将磁信号调制到高频,有效避开低频1/f噪声影响。设计一种实现电/磁转换功能的微机械结构,利用TMR电阻构成惠斯通电桥测量磁信号,实现海洋电场的高水平低噪声检测。仿真与实验结果在1 Hz处噪声分别达到1.16 nV/(Hz)~(1/2)和1.41 nV/(Hz)~(1/2),优于传统电测量方案的噪声水平,验证该方法的有效性。     

A new magnetic alloy of high constant permeability

Several conventional alloys of constant permeability used previously were of lower values of permeability, or they would change obviously in different intensities of magnetic field and thus could not satisfy some specific requirements of electrical application. The alloys consisted of Ni 60-70 wt%, Fe 29-39wt% and Mn 0-1 wt% were investigated. The best of those alloys for certain use is 1J66 alloy in China. It is a new alloy of high constant permeability with the chemical composition as Fe 34 wt%, Ni 65 wt% and Mn 1 wt%. Its AC induction permeabilitymu_{L}= 3400G/Oe (f=60Hz). Within the range of Bm=20-6800 G, the stability of AC induction permeability (f=60Hz)alpha_{sim} = 4.3%. The α is defined asalpha = mumax-mumin/mumin,alphamaxandmuminare the maximum and the minimum of permeability respectively in the range of given magnetic field. The temperature stability αTof μLwithin the range of temperature from -60°C to +90°C is 2.9%. The saturation magnetic induction of 1J66 alloy equals 13500 G. The remanenceB_{r} < 150G. The coercive forceH_{c} = 0.05Oe. The oxygen content of 0.015- 0.04% in the alloy is considered necessary for obtaining a good stability of permeability.