Detection Of Thyroid Stimulating Hormone Research Articles

Bridging the Gap between Digital Assays and Point-of-Care Testing: Automated, Low Cost, and Ultrasensitive Detection of Thyroid Stimulating Hormone.

Medical diagnostics is moving toward disease-related target detection at very low concentrations because of the (1) quest for early-stage diagnosis, at a point where only limited target amounts are present, (2) trend toward minimally invasive sample extraction, yielding samples containing low concentrations of target, and (3) need for straightforward sample collection, usually resulting in limited volume collected. Hence, diagnostic tools allowing ultrasensitive target detection at the point-of-care (POC) are crucial for simplified and timely diagnosis of many illnesses. Therefore, we developed an innovative, fully integrated, semi-automated, and economically viable platform based on (1) digital microfluidics (DMF), enabling automated manipulation and analysis of very low sample volumes and (2) low-cost disposable DMF chips with microwell arrays, fabricated via roll-to-roll processes and allowing digital target counting. Thyroid stimulating hormone detection was chosen as a relevant application to show the potential of the system. The assay buffer was selected using design of experiments, and the assay was optimized in terms of reagent concentration and incubation time toward maximum sensitivity. The hydrophobic-in-hydrophobic microwells showed an unparalleled seeding efficiency of 97.6% ± 0.6%. A calculated LOD of 0.0013 μIU/mL was obtained, showing the great potential of the platform, especially taking into account the very low sample volume analyzed (1.1 μL). Although validation (in biological matrix) and industrialization (full automation) steps still need to be taken, it is clear that the combination of DMF, low-cost DMF chips, and digital analyte counting in microwell arrays enables the implementation of ultrasensitive and reliable target detection at the POC.

High-Sensitive Detection and Quantitative Analysis of Thyroid-Stimulating Hormone Using Gold-Nanoshell-Based Lateral Flow Immunoassay Device.

Au nanoparticles (AuNPs) have been used as signal reporters in colorimetric lateral flow immunoassays (LFAs) for decades. However, it remains a major challenge to significantly improve the detection sensitivity of traditional LFAs due to the low brightness of AuNPs. As an alternative approach, we overcome this problem by utilizing 150 nm gold nanoshells (AuNSs) that were engineered by coating low-density silica nanoparticles with a thin layer of gold. AuNSs are dark green, have 14 times larger surface area, and are approximately 35 times brighter compared to AuNPs. In this study, we used detection of thyroid-stimulating hormone (TSH) in a proof-of-concept assay. The limit of detection (LOD) with AuNS-based LFA was 0.16 µIU/mL, which is 26 times more sensitive than the conventional colorimetric LFA that utilizes AuNP as a label. The dynamic range of the calibration curve was 0.16–9.5 µIU/mL, making it possible to diagnose both hyperthyroidism (<0.5 µIU/mL) and hypothyroidism (>5 µIU/mL) using AuNS-based LFA. Thus, the developed device has a strong potential for early screening and diagnosis of diseases related to the thyroid hormone.

Sensitive detection of thyroid stimulating hormone by inkjet printed microchip with a double signal amplification strategy

An electrochemical immunosensor for sensitive detection of thyroid stimulating hormone (TSH) has been developed by using an inkjet printed microchip and based on a double signal amplification strategy using magnetic beads (MBs), alkaline phosphatase (ALP) and p-aminophenyl phosphate (pAPP) reaction. Differential pulse voltammetry (DPV), cyclic voltammogram (CV) and amperometric i–t curve (i–t) were employed to characterize the immunosensor. High sensitivity and good selectivity were observed. The detection linear range was from 0.01μIU/mL to 10μIU/mL, in which the peak currents increased along with the concentration. The detection limit was 0.005μIU/mL at S/N=3. The immunosensor was also applied for TSH detection in human serum with recoveries from 98.0% to 101.8% and relative standard deviations from 1.3% to 3.1%, demonstrating potential value in clinical diagnosis.

Measurement of thermally stimulated discharge current (TSDC) for detection of thyroid-stimulating hormone (TSH) in human blood

TSDC was measured in human blood sample with different level of TSH. The origin of TSDC could be explained on the basis of bioelectret effect in biological sample, which is interpreted by fundamental concepts of bioelectret state in biopolymers. The concept of thermal analysis of blood gives the basis of TSDC peaks. The nature of the TSDC peaks and their relation to the physicochemical transformations and the interphase interactions in these systems are discussed. It is observed that the position of TSDC peak is different for different level of TSH. The activation energy, charge released, and peak current were observed to decrease with TSH contents in human blood. The increase of TSH nonpolar salt in human blood is associated with the decrease of charge carriers due to denaturation or hydration of protein with temperature. It is noted that TSDC can be suitable diagnostics of TSH in human blood.

Application of Eu(III) nanoparticle labels in time-resolved phosphorescence analysis for detection of thyroid stimulating hormone

Eu(III) chelate-bonded polymer nanoparticles were used as phosphorescent labels to develop a highly sensitive phosphorescence analysis (PHOSPHANTM) for detection of human thyroid stimulating hormone (TSH). The phosphorescence was recorded in a time-resolved mode from microzones (microarrays) that were printed on the bottoms of the wells of standard polystyrene microplates. TSH was detected in paper discs (3.2 mm in diameter) that were punched from capillary blood that was dried on filter paper. The basic analytical and functional characteristics of this new method were similar to those of the commercial reagent kit for TSH detection in dried blood spots from newborns. Both methods made it possible to correctly detect the hormone concentration in the range of 0–250 μIU/mL blood. The analytical sensitivity of the Eu(III) nanoparticle label was 0.46 ± 0.1 μIU/mL (corresponded to approximately 17 fmol/L), which was comparable with the most sensitive assays. The matrix effect was insignificant. The coefficient of the variation of the results (up to 20%) was higher than in the commercial kit. This highly sensitive new test system could be integrated into PHOSPHAN-based multiplex tests to aid in simultaneous detection of markers for several congenital disorders in newborns.

Bioassays for TSH Receptor Autoantibodies, from FRTL-5 Cells to TSH Receptor-LH/CG Receptor Chimeras: The Contribution of Leonard D. Kohn.

Since the discovery 60 years ago of the “long-acting thyroid stimulator” by Adams and Purves, great progress has been made in the detection of thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) in Graves’ disease. Today, commercial assays are available that can detect TRAbs with high accuracy and provide diagnostic and prognostic evaluation of patients with Graves’ disease. The present review focuses on the development of TRAbs bioassays, and particularly on the role that Leonard D. Kohn had in this. Indeed, 30 years ago, the Kohn group developed a bioassay based on the use of FRTL-5 cells that was characterized by high reproducibility, feasibility, and diagnostic accuracy. Using this FRTL-5 bioassay, Kohn and his colleagues were the first to develop monoclonal antibodies (moAbs) against the TSHR. Furthermore, they demonstrated the multifaceted functional nature of TRAbs in patients with Graves’ disease, with the identification of stimulating and blocking TRAbs, and even antibodies that activated pathways other than cAMP. After the cloning of the TSHR, the Kohn laboratory constructed human TSHR–rat luteinizing hormone/chorionic gonadotropin receptor chimeras. This paved the way to a new bioassay based on the use of non-thyroid cells transfected with the Mc4 chimera. The new Mc4 bioassay is characterized by high diagnostic and prognostic accuracy, greater than for other assays. The availability of a commercial kit based on the Mc4 chimera is spreading the use of this assay worldwide, indicating its benefits for these patients with Graves’ disease. This review also describes the main contributions made by other researchers in TSHR molecular biology and TRAbs assay, especially with the development of highly potent moAbs. A comparison of the diagnostic accuracies of the main TRAbs assays, as both immunoassays and bioassays, is also provided.

Self-driven filter-based blood plasma separator microfluidic chip for point-of-care testing

There is currently a growing need for lab-on-a-chip devices for use in clinical analysis and diagnostics, especially in the area of patient care. The first step in most blood assays is plasma extraction from whole blood. This paper presents a novel, self-driven blood plasma separation microfluidic chip, which can extract more than 0.1 μl plasma from a single droplet of undiluted fresh human blood (∼5 μl). This volume of blood plasma is extracted from whole blood with high purity (more than 98%) in a reasonable time frame (3 to 5 min), and without the need for any external force. This would be the first step towards the realization of a single-use, self-blood test that does not require any external force or power source to deliver and analyze a fresh whole-blood sample, in contrast to the existing time-consuming conventional blood analysis. The prototypes are manufactured in polydimethylsiloxane that has been modified with a strong nonionic surfactant (Silwet L-77) to achieve hydrophilic behavior. The main advantage of this microfluidic chip design is the clogging delay in the filtration area, which results in an increased amount of extracted plasma (0.1 μl). Moreover, the plasma can be collected in one or more 10 μm-deep channels to facilitate the detection and readout of multiple blood assays. This high volume of extracted plasma is achieved thanks to a novel design that combines maximum pumping efficiency without disturbing the red blood cells’ trajectory through the use of different hydrodynamic principles, such as a constriction effect and a symmetrical filtration mode. To demonstrate the microfluidic chip’s functionality, we designed and fabricated a novel hybrid microdevice that exhibits the benefits of both microfluidics and lateral flow immunochromatographic tests. The performance of the presented hybrid microdevice is validated using rapid detection of thyroid stimulating hormone within a single droplet of whole blood.

An innovative sample-to-answer polymer lab-on-a-chip with on-chip reservoirs for the POCT of thyroid stimulating hormone (TSH)

A new sample-to-answer polymer lab-on-a-chip, which can perform immunoassay with minimum user intervention through on-chip reservoirs for reagents and single-channel assay system, has been designed, developed and successfully characterized as a point-of-care testing (POCT) cartridge for the detection of thyroid stimulating hormone (TSH). Test results were obtained within 30 minutes after a sample was dropped into the POCT cartridge. The analyzed results of TSH showed a linear range of up to 55 μIU mL(-1) with the limit of detection (LOD) of 1.9 μIU mL(-1) at the signal-to-noise ratio (SNR) of 3. The reagents stored in the on-chip reservoirs maintained more than 97% of their initial volume for 120 days of storage time while the detection antibody retained its activity above 98% for 120 days. The sample-to-answer polymer lab-on-a-chip developed in this work using the mass-producible and low-cost polymer is well suited for the point-of-care testing of rapid in vitro diagnostics (IVD) of TSH.

Sensitivity Enhancement of Optical Immunosensors with Nanoparticles

In recent years, several optical sensor techniques have been developed for the direct monitoring of biomolecular recognition processes at the surface of a sensor chip. Applications of these immunosensors for the determination of substances in serum could be demonstrated only for a few analytes due to the lack of sensitivity. Beside nonspecific binding of serum components to the sensor surface, the analytical sensitivity of these sensors is limited by the molecular weight of the analyte, so that smaller analyte molecules give only a moderate sensor response. In order to enhance the sensor signal, the use of mass labels, such as latex particles, was proposed in the literature. However, detection limits comparable to those of conventional ELISA techniques could not be realized so far.We demonstrate the optimization of a “nanoparticle enhanced immunosensor assay” for the detection of thyroid stimulating hormone, with respect to the particle coating, size, and nonspecific binding. The developed prototype assay requires a sample volume of 225 μL and has a measuring range up to 35 mIU/L. For the first time, we obtained a detection limit of 0.03 mIU/L (0.1 pm), which is fully competitive to conventional ELISA techniques. The assay allows serum samples to be measured with good precision and dilution linearity. The sensor can be reused several times and shows an excellent correlation to a commercial enzyme immunoassay.

Simultaneous detection of thyroid stimulating hormone (TSH) and long acting thyroid stimulator (LATS)

We have developed a highly sensitive assay for detecting TSH in human serum, capable of measuring as little as 1×10 −5 mU/ml. With this technic we can demonstrate TSH in sera from all normal subjects as well as in patients with hypopituitarism and Graves' disease where it has not previously been measurable. High levels are found in patients with myxedema. In addition, we are able to simultaneously demonstrate in sera from the majority of patients with Graves' disease a 7S gamma globulin with an affinity for the thyroid cell cytoplasm. The antigen for this substance is not destroyed by polar solvents, its presence is not correlatable with thyroid antibodies, and it occurs independent of the presence of infiltrative exophthalmos or pretibial myxedema. The presence of this material can be correlated with long acting thyroid stimulator activity in the McKenzie assay and appears to be related to or identical with LATS.

The simultaneous detection of thyroid and thyrotrophic hormones in vertebrate sera.

Although it is generally accepted that reciprocal regulatory control exists between the thyroid and pituitary glands (Aron, 1930; Loeb, Bassett, and Friedman, 1930; Houssay, Biasotti, and Magdalena, 1932; Kuschinsky, 1933; VanEck, 1939; Uotila, 1940; Galli-Mainini, 1941; Rawson and colleagues, 1942, 1943) the inadequacy of present methods for the detection of thyroid stimulating hormone (TSH) in the circulating fluids continues to thwart complete understanding of anterior-pituitary-thyroid gland interaction in the vertebrate organism. Assays of pituitary thyrotrophin have been done extensively and with reasonably consistent results (Adams, 1946), nevertheless, these give no measure of the amounts of TSH released into the circulation. Aside from the recent reports of DeRobertis (1948) and Purves and Griesbach (1949), there is little information available which accurately defines the levels of thyrotrophin in blood in either normal, experimental, or clinical conditions. Bioassays utilizing treated as well a...