Analyzer-to-Analyzer Variations in Assaying Ultralow Concentrated Biomarkers Associated with Neurodegenerative Diseases Using Immunomagnetic Reduction.

By utilizing a high-temperature superconducting quantum interference device (high-Tc SQUID) magnetometer, an alternating current (AC) magnetosusceptometer, referred to as an analyzer, was developed for ultrasensitive immunoassays. The analyzer has been applied to assay biomarkers in human plasma associated with Alzheimer's disease (AD) and Parkinson's disease (PD). The involved assay methodology is the so-called immunomagnetic reduction (IMR). Such an analyzer has been approved for clinical use in Taiwan and Europe. The mass production of the analyzer is needed for clinical utilities. The issue of exploring analyzer-to-analyzer variations in the performances becomes critical. Unfortunately, there is no standard characterization to determine the variations in performances among analyzers. In this study, key characterizations, such as output signal stability, signal-to-noise ratio, measured concentrations of a control sample, etc., are proposed. In total, three analyzers are characterized in this work. The detected biomarkers include amyloid peptides, total tau protein, phosphorylated tau protein, and α-synuclein protein for AD and PD. Through one-way ANOVA for any of the characterizations among the three analyzers, it was found that there was no significant difference in any of these characterizations among the analyzers (p > 0.05). Furthermore, the three analyzers are applied to assay biomolecules for AD and PD in reference samples. High correlations (r > 0.8) in measured concentrations of any of these biomarkers in reference samples were obtained among the three analyzers. The results demonstrate that the proposed characterizations are feasible for achieving consistent performance among high-Tc SQUID-based AC magnetosusceptometers for assaying biomolecules.

Effect of Time to Detection on the Measured Concentrations of Blood Proteins Associated with Alzheimer's Disease.

Background: For assays using immunomagnetic reduction, a reagent composed of antibody-functionalized magnetic nanoparticles is dispersed in phosphate-buffered saline solution. The real-time signals of alternating-current (ac) magnetic susceptibility, χac, of the reagent are subsequently recorded after mixing the reagent with a biofluid sample. After mixing the reagent and sample, the reduction in χac of the mixture is calculated and used to quantify the concentration of the target biomarker in the sample. The reduction does not occur immediately but rather occurs at some time after mixing. This observation implies that the time elapsed before recording the real-time signals of χac of a reagent-sample mixture needs to be investigated to ensure that the signals are fully recorded. In this work, the effect of time to detection on the measured concentrations of proteins in human plasma after mixing the reagent and sample is examined. Methods: The proteins analyzed are related to Alzheimer's disease: amyloid ß 1-40, amyloid ß 1-42, and Tau protein. The investigated times to detection after the mixing the reagent and sample are 0, 20, 30, 40, and 120 min. Results: The results show that the recording of real-time signals of χac should be conducted within 20 min after mixing the reagent and sample.

Development of a 36-Channel Instrument for Assaying Biomarkers of Ultralow Concentrations Utilizing Immunomagnetic Reduction.

With the demands of the high-throughput assay of biomarkers of ultralow concentrations in clinics, a 36-channel instrument utilizing immunomagnetic reduction (IMR) has been developed. The instrument involves the use of a high-T c superconducting-quantum-interference-device (SQUID) magnetometer to detect the signals due to the associations between target biomarker molecules and the antibody-functionalized magnetic nanoparticles in the reagent of IMR. In addition to illustrating the design and the measurements of the instrument, the assay characterizations for eight kinds of biomarkers related to neurodegenerative disease are investigated. Furthermore, the assay results among three independent instruments were compared. For an instrument, the channel-to-channel variations in measured concentrations of biomarkers are within a range of 2.09 to 5.62%. The assay accuracy was found to be from 99 to 103.7%. The p values in measured concentrations for any of the tested biomarkers were higher than 0.05 among the three instruments. The results demonstrate high throughput, high stability, and high consistency for the SQUID-IMR instruments.

Investigation of the Number of Tests Required for Assaying Plasma Biomarkers Associated with Alzheimer's Disease Using Immunomagnetic Reduction.

INTRODUCTION: Concentrations of plasma biomarkers associated with Alzheimer's disease have been reported to be as low as several tens of picograms/milliliter (pg/ml). However, in assays measuring these biomarkers, it is likely that repeated measurements are necessary to obtain reliable values. METHODS: We performed assays as a single test or as duplicate, quadruplicate, fivefold and tenfold repeated tests, on samples spiked with different concentrations of amyloid ß 1-40 (Aß1-40; 1-1000 pg/ml), Aß1-42 (1-30,000 pg/ml) and total Tau protein (T-Tau; 0.1-1000 pg/ml), with the aim to to calculate the coefficients of variation (CVs). RESULTS: The results demonstrated common changes in the CVs with changes in the number of tests for a given sample: the CVs decreased with increases in the number of tests from one to ten. All CV values were distributed within the range of 0.35 to 15.5%; as such, the CV values were all lower than the acceptable value of 20%. CONCLUSION: Based on this study, a single assay of Aß1-40, Aß1-42 and T-Tau, respectively, provides reliable results in terms of the measurement of that plasma biomarker.

An Automatic Embedded Device Registration Procedure Based on the OGC SensorThings API.

Sensor Web and Internet of Things (IoT) (SW-IoT) have been attracting attention from various fields. Both of them deploy networks of embedded devices to monitor physical properties (i.e., sensing capability) or to be controlled (i.e., tasking capability). One of the most important tasks to realize the SW-IoT vision is to establish an open and interoperable architecture, across the device layer, gateway layer, service layer, and application layer. To achieve this objective, many organizations and alliances propose standards for different layers. Among the standards, Open Geospatial Consortium (OGC) SensorThings API is arguably one of the most complete and flexible service standards. However, the SensorThings API only address heterogeneity issues in the service layer. Embedded devices following proprietary protocols need to join closed ecosystems and then link to the SensorThings API ecosystem via customized connectors. To address this issue, one could first follow another device layer and gateway layer open standards and then perform data model mapping with the SensorThings API. However, the data model mapping is not always straightforward as the standards were designed independently. Therefore, this research tries to propose a more direct solution to unify the entire SW-IoT architecture by extending the SensorThings API ecosystem to the gateway layer and the device layer. To be specific, this research proposes SW-IoT Plug and Play (IoT-PNP) to achieve an automatic registration procedure for embedded devices. The IoT-PNP contains three main components: (1) A description file describing device metadata and capabilities, (2) a communication protocol between the gateway layer and the device layer for establishing connections, and (3) an automatic registration procedure for both sensing and tasking capabilities. Overall, we believe the proposed solution could help achieve an open and interoperable SW-IoT end-to-end architecture based on the OGC SensorThings API.

Plasma Biomarkers Differentiate Parkinson's Disease From Atypical Parkinsonism Syndromes.

Objective: Parkinson's disease (PD) has significant clinical overlaps with atypical parkinsonism syndromes (APS), which have a poorer treatment response and a more aggressive course than PD. We aimed to identify plasma biomarkers to differentiate PD from APS. Methods: Plasma samples (n = 204) were obtained from healthy controls and from patients with PD, dementia with Lewy bodies (DLB), multiple system atrophy, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), or frontotemporal dementia (FTD) with parkinsonism (FTD-P) or without parkinsonism. We measured plasma levels of α-synuclein, total tau, p-Tau181, and amyloid beta 42 (Aß42) by immunomagnetic reduction-based immunoassay. Results: Plasma α-synuclein level was significantly increased in patients with PD and APS when compared with controls and FTD without parkinsonism (p < 0.01). Total tau and p-Tau181 were significantly increased in all disease groups compared to controls, especially in patients with FTD (p < 0.01). A multivariate and receiver operating characteristic curve analysis revealed that a cut-off value for Aß42 multiplied by p-Tau181 for discriminating patients with FTD from patients with PD and APS was 92.66 (pg/ml)2, with an area under the curve (AUC) of 0.932. An α-synuclein cut-off of 0.1977 pg/ml could separate FTD-P from FTD without parkinsonism (AUC 0.947). In patients with predominant parkinsonism, an α-synuclein cut-off of 1.388 pg/ml differentiated patients with PD from those with APS (AUC 0.87). Conclusion: Our results suggest that integrated plasma biomarkers improve the differential diagnosis of PD from APS (PSP, CBD, DLB, and FTD-P).

Analytical performance of reagent for assaying tau protein in human plasma and feasibility study screening neurodegenerative diseases.

Immunomagnetic reduction (IMR), which involves the use of antibody-functionalized magnetic nanoparticles to specifically label target biomarkers, was utilized to develop an assay for total tau protein in human plasma. The analytic properties of the IMR assay on tau protein were investigated. The limit of detection was found to be 0.026 pg/ml. Other properties such as Hook effect, assay linearity, dilution recovery range, reagent stability, interference test, and spiked recovery were also characterized. The ultra-sensitive IMR assay was applied to detect the plasma tau protein levels of subjects with prevalent neurodegenerative diseases, such as Alzheimer's disease (AD), mild cognitive impairment (MCI) due to AD, Parkinson's disease (PD), frontotemporal dementia (FTD) and vascular dementia (VD). The concentrations of plasma tau protein in patients with VD, PD, MCI due to AD, FTD, and AD patients were higher than that of healthy controls. Using an ROC curve analysis, the cutoff value for discriminating dementia patients from healthy controls was 17.43 pg/ml, resulting in 0.856 and 0.727 for clinical sensitivity and specificity, respectively. The area under the ROC curve was 0.908. These results imply that the IMR plasma tau assay would be useful to screen for prevalent neurodegenerative diseases.

Plasma α-synuclein predicts cognitive decline in Parkinson's disease.

OBJECTIVE: α-Synuclein is critical to the pathogenesis of Parkinson's disease (PD). Few studies examined the plasma levels of α-synuclein due to the exceptionally low level of α-synuclein in plasma compared with cerebrospinal fluid. We aimed to investigate plasma α-synuclein in patients with PD of different disease severity. METHODS: There were total 114 participants, including 80 patients with PD and 34 controls, in the study. Participants received a complete evaluation of motor and non-motor symptoms, including cognitive function. We applied immunomagnetic reduction-based immunoassay to measure plasma levels of α-synuclein. RESULTS: Plasma levels of α-synuclein were significantly higher in patients with PD compared with controls (median: 1.56 pg/mL, 95% CI 1.02 to 1.98 pg/mL vs 0.02 pg/mL, 95% CI 0.01 to 0.03 pg/mL; p<0.0001). Although there was a significant increase in plasma α-synuclein levels in PD patients with a higher Hoehn-Yahr (H-Y) stage, there was no correlation with motor symptom severity, as assessed by Unified Parkinson's Disease Rating Scale part III scores, after confounders (age, gender, and disease duration) were taken into account. However, plasma α-synuclein levels were significantly higher in PD patients with dementia (PDD) than in PD patients with mild cognitive impairment (PD-MCI) or normal cognition (0.42 pg/mL, (95% CI 0.25 to 0.93) for PD with normal cognition; 1.29 pg/mL (95% CI 0.76 to 1.93) for PD-MCI and 4.09 pg/mL (95% CI 1.99 to 6.19) for PDD, p<0.01) and were negatively correlated with Mini-Mental State Examination scores (R2-adjusted=0.3004, p<0.001), even after confounder adjustment. CONCLUSIONS: Our data suggest that plasma α-synuclein level correlates with cognitive decline but not motor severity in patients with PD. Plasma α-synuclein could serve as a surrogate biomarker for patients at risk of cognitive decline.

Substrate-induced structures of bismuth adsorption on graphene: a first principles study.

The geometric and electronic properties of Bi-adsorbed monolayer graphene, enriched by the strong effect of a substrate, are investigated by first-principles calculations. The six-layered substrate, corrugated buffer layer, and slightly deformed monolayer graphene are all simulated. Adatom arrangements are thoroughly studied by analyzing the ground-state energies, bismuth adsorption energies, and Bi-Bi interaction energies of different adatom heights, inter-adatom distance, adsorption sites, and hexagonal positions. A hexagonal array of Bi atoms is dominated by the interactions between the buffer layer and the monolayer graphene. An increase in temperature can overcome a ∼50 meV energy barrier and induce triangular and rectangular nanoclusters. The most stable and metastable structures agree with the scanning tunneling microscopy measurements. The density of states exhibits a finite value at the Fermi level, a dip at ∼-0.2 eV, and a peak at ∼-0.6 eV, as observed in the experimental measurements of the tunneling conductance.

Development of an ultra-high sensitive immunoassay with plasma biomarker for differentiating Parkinson disease dementia from Parkinson disease using antibody functionalized magnetic nanoparticles.

BACKGROUND: It is difficult to discriminate healthy subjects and patients with Parkinson disease (PD) or Parkinson disease dementia (PDD) by assaying plasma α-synuclein because the concentrations of circulating α-synuclein in the blood are almost the same as the low-detection limit using current immunoassays, such as enzyme-linked immunosorbent assay. In this work, an ultra-sensitive immunoassay utilizing immunomagnetic reduction (IMR) is developed. The reagent for IMR consists of magnetic nanoparticles functionalized with antibodies against α-synuclein and dispersed in pH-7.2 phosphate-buffered saline. A high-Tc superconducting-quantum-interference-device (SQUID) alternative-current magnetosusceptometer is used to measure the IMR signal of the reagent due to the association between magnetic nanoparticles and α-synuclein molecules. RESULTS: According to the experimental α-synuclein concentration dependent IMR signal, the low-detection limit is 0.3 fg/ml and the dynamic range is 310 pg/ml. The preliminary results show the plasma α-synuclein for PD patients distributes from 6 to 30 fg/ml. For PDD patients, the concentration of plasma α-synuclein varies from 0.1 to 100 pg/ml. Whereas the concentration of plasma α-synuclein for healthy subjects is significantly lower than that of PD patients. CONCLUSIONS: The ultra-sensitive IMR by utilizing antibody-functionalized magnetic nanoparticles and high-Tc SQUID magnetometer is promising as a method to assay plasma α-synuclein, which is a potential biomarker for discriminating patients with PD or PDD.

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.

Discriminating hepatocellular carcinoma in rats using a high-Tc SQUID detected nuclear resonance spectrometer in a magnetic shielding box.

In this study, we report the spin-lattice relaxation rate of hepatocellular carcinoma (HCC) and normal liver tissue in rats using a high-T(c) superconducting quantum interference device (SQUID) based nuclear magnetic resonance (NMR) spectrometer. The resonance spectrometer used for discriminating liver tumors in rats via the difference in longitudinal relaxation time in low magnetic fields was set up in a compact and portable magnetic shielding box. The frequency-domain NMR signals of HCC tissues and normal liver tissues were analyzed to study their respective longitudinal relaxation rate T(1) (-1). The T(1) (-1) of liver tissues for ten normal rats and ten cancerous rats were investigated respectively. The averaged T(1) (-1) value of normal liver tissue was (6.41±0.66) s(-1), and the averaged T(1) (-1) value of cancerous tissue was (3.38±0.15) s(-1). The ratio of T(1) (-1) for normal liver tissues and cancerous liver tissues of the rats investigated is estimated to be 1.9. Since this significant statistical difference, the T(1) (-1) value can be used to distinguish the HCC tissues from normal liver tissues. This method of examining liver and tumor tissues has the advantages of being convenient, easy to operate, and stable.

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.

A study of J-coupling spectroscopy using the Earth's field nuclear magnetic resonance inside a laboratory.

In this paper, an instrumentation of the Earth's field nuclear magnetic resonance (EFNMR) inside a laboratory is presented. A lock-in analysis (LIA) technique was proposed to enhance the signal-to-noise ratio (SNR). A SNR of 137.8 was achieved in a single measurement for 9 ml tap water, and the LIA technique significantly enhanced the SNR to 188 after a 10-average in a noisy laboratory environment. The proton-phosphorus coupling in trimethyl phosphate ((CH(3)O)(3)PO) with J-coupling J[H,F]=(10.99±0.013) Hz has been demonstrated. The LIA technique improves the SNR, and a 2.6-fold improvement in SNR over that of the frequency-adjusted averaging is achieved. To reduce the noise in EFNMR, it was suggested that the LIA technique and the first order gradient shim be used to achieve a subhertz linewidth.