Zinc oxide nanoparticles based microfluidic immunosensor applied in congenital hypothyroidism screening

In this article, we present an innovative approach for congenital hypothyroidism (CHT) screening. This pathology is the most common preventable cause of mental retardation, affecting newborns around the world. Its consequences could be avoided with an early diagnosis through the thyrotropin (TSH) level measurement. To accomplish the determination of TSH, synthesized zinc oxide (ZnO) nanobeads (NBs) covered by chitosan (CH), ZnO-CH NBs, were covalently attached to the central channel of the designed microfluidic device. These beads were employed as platform for anti-TSH monoclonal antibody immobilization to specifically recognize and capture TSH in neonatal samples without any special pretreatment. Afterwards, the amount of this trapped hormone was quantified by horseradish peroxidase (HRP)-conjugated anti-TSH antibody. HRP reacted with its enzymatic substrate in a redox process, which resulted in the appearance of a current whose magnitude was directly proportional to the level of TSH in the neonatal sample. The structure and morphology of synthesized ZnO-CH NBs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The calculated detection limits for electrochemical detection and the enzyme-linked immunosorbent assay procedure were 0.00087 μUI mL^?1 and 0.015 μUI mL^?1, respectively, and the within- and between-assay coefficients of variation were below 6.31 % for the proposed method. According to the cut-off value for TSH neonatal screening, a reasonably good limit of detection was achieved. These above-mentioned features make the system advantageous for routine clinical analysis adaptation.     

浙江省建立新生儿TSH血样采集邮寄检验服务网络的策略与评价

为研究新生儿促甲状腺激素（ＴＳＨ）血样采集方法,建立了采集邮寄服务网络,提高ＴＳＨ检验工作水平和覆盖率。结果表明,发挥网络功能对开展新生儿ＴＳＨ检验工作是非常重要的。     

Enzyme Immunoassay of Thyroid-Stimulating Hormone Using Dried Blood Samples a Simple Technique of Screening for Congenital Hypothyroidism

Abstract

A method for enzyme imnunoassay of thyroid-stimulating hormone (TSH) in dried blood spotted onto filter paper has been developed. TSH was conjugated to horse-radish peroxidase according to Nakane's method. Separation of the bound and free fractions was obtained by a double antibody solid phase method using polyacetal beads which were coated with the purified IgG fraction from goat anti-rabbit IgG serum. p-Hydroxyphenyl propionic acid was used as substrate for the fluorophotometric assay of peroxidase activity. The assay sensitivity is 0.07, μU TSH/assay tube, which is equivalent to μU/ml when five 3 mm discs of dried blood spot are assayed. TSH values in dried blood samples obtained by this method correlate well with those of serum samples obtained by radioimmunoassay (r=0.89). The coefficients of variation were 6.8 to 13.4% (within assay) and 5 to 40% (between assay). The enzyme immunoassay of TSH presented here is applicable to the mass-screening for congenital hypothyroidism of neonate.     

Fundamental studies on thyroid stimulating hormone immunoradiometric assay kit (TSH RIABEAD II)

We have reported fundamental studies on the TSH immunoradiometric assay, using TSH RIABEAD II kit (Dainabot). The sensitivity of the assay was 0.03 mu IU/ml and its C.V. was 27.2%. Intra- and inter-assay C.V. were less than 5%. Dilution test and recovery test were good. Serum TSH level was 0.3-4.0 mu IU/ml in normal subjects, less than 0.03 mu IU/ml in untreated Graves' disease and subacute thyroiditis. Therefore, it was found that the clear difference exist in serum TSH levels between normal subjects and patients with untreated Graves' disease. There was a well correlation on the serum TSH levels between this method and TSH radioimmunoassay kit (Amerlex TSH, r = 0.983). Especially, the measurement of serum TSH levels, using immunoradiometric assay kit, was useful for the diagnosis of patients with Graves' disease.     

Immunoradiometric assay for the in-vitro determination of thyroid stimulating hormone in human serum and plasma using solid phase anti-TSH cellulose particles

Summary Development, optimization and validation of immunoradiometric assay (IRMA) using solid phase-cellulose particles for the measurement of TSH in human serum or plasma are described. The preparation of ¹²⁵I anti-TSH monoclonal antibody (MoAb) was carried out by two different methods (Chloramine-T and Iodogen). It was found that Ch-T method gave approximately the same results as the iodogen method. The activation of cellulose particles using 1,1-carbonyl diimidazole (CDI) and coupling of these solid phase particles with sheep anti-TSH were carried out. Optimization and validation of the assay were undertaken. The reproducibility as measured by the intra- and inter-assay variations is acceptable. The recovery and dilution tests indicated accurate calibration and appropriate matrix. No significant position effect was recognized. The different modes of sampling tested did not affect significantly the results of the present study. The present technique agreed well with the currently used commercial kit (DPC, IRMA). The cellulose particles of the present system for estimation of TSH retain their characteristics during storage for 6 months at 4 °C. In conclusion, this technique proved to be sensitive, specific, precise and accurate for routine laboratory use.     

Quantitative determination of norepinephrine and epinephrine in human urine by HPLC with electrochemical detection

Conclusion HPLC-ED proved to be a practicable method for the differential assay of urinary NE and E with high precision, accuracy and specificity; for clinical laboratories it should be considered as control method and alternative for traditional fluorescence assays. The method can be easily extended to the simultaneous determination of urinary dopamine.

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Quantitative Bestimmung von Noradrenalin und Adrenalin im menschlichen Urin mit Hilfe der HPLC und elektrochemischer Detektion

     

Spectrophotometric determination of some phenols using m-Phenylenediamine and sodium metaperiodate

Summary A new spectrophotometric method for the assay of catechol, guaiacol,o-aminophenol,p-aminophenol and metol using them-phenylenediamine —IO₄-reagent has been developed. The method is simple and accurate within ±1.0%. It has been applied to the estimations of paracetamol through its hydrolysed product and metol in photographic developers.

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Spektrophotometrische Bestimmung einiger Phenole mit m-Phenylendiamin und Natrium-Metaperjodat

Zusammenfassung Ein neues spcktrophotometrisches Verfahren zur Bestimmung von Brenzcatechin, Guajakol,o-Aminophenol und Metol mit Hilfe vonm-Phenylendiamin und Perjodat wurde ausgearbeitet. Es ist einfach und innerhalb ±1,0% genau und wurde zur Bestimmung von Paracetamol über sein Hydrolyseprodukt und Metol in photographischen Entwicklern verwendet.

     

Nanogold–polyaniline–nanogold microspheres-functionalized molecular tags for sensitive electrochemical immunoassay of thyroid-stimulating hormone

Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold–polyaniline–nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab₂) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L⁻¹ H₂O₂ by using GPG-labeled HRP-Ab₂ as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab₂. Several labeling protocols comprising HRP-Ab₂, nanogold particle-labeled HRP-Ab₂, and polyaniline nanospheres-labeled HRP-Ab₂, were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab₂. With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab₂ to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 μIU mL⁻¹ with a detection limit (LOD) of 0.005 μIU mL⁻¹ TSH at the 3s_B criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH.     

Thermometric assay of some aldoses

Summary A method is proposed for the determination of milligram amounts of some aldohexoses and aldopentoses. After oxidation using an excess of periodate in acidic solution, the unreacted periodate is determined thermometrically by titration with mannitol, which gives reaction products which cannot react with those already produced. This removes the possibility of errors caused by interaction of the products of the oxidation reaction and the assay reaction. The method gives results as acceptable as those obtained by the Malaprade method, in a shorter time and with better sensitivity, and can be used in industrial samples where methods involving visual detection of the end point cannot be used.

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Thermometrische Bestimmung einiger Aldosen

Zusammenfassung Ein Verfahren zur Bestimmung von mg-Mengen einiger Aldohexosen und -pentosen wird vorgeschlagen. Nach Oxydation mit überschüssigem Perjodat in saurer Lösung wird Perjodat thermometrisch mit Mannit zurücktitriert. Hierbei entstehen Reaktionsprodukte, die mit den bereits gebildeten nicht reagieren. Dadurch werden entsprechende Fehlermöglichkeiten ausgeschaltet. Die erhaltenen Ergebnisse sind denen der Malaprade-Methode vergleichbar; das Verfahren bietet jedoch den Vorteil geringeren Zeitbedarfs und größerer Empfindlichkeit. Es kann für industrielle Proben verwendet werden, bei denen visuelle Endpunktsindikation nicht möglich ist.

     

Analysis of high-purity water: a direct determination of ultratrace inorganic impurities by monostandard neutron activation analysis

Summary A direct method for determining ultratrace inorganic impurities in high-purity water is presented, which consists in activation of a large volume of water (250 ml) by reactor neutron activation, subsequent gamma spectrometric assay and concentration evaluation by the monostandard procedure. The method requires neither preconcentration nor postconcentration and provides a sensitivity for a large number of elements in the concentration range of parts per trillion (ppt: 10^–12 g/g) or even much less. Using the method, over twenty different ultratrace elements are quantitatively determined in two different high-purity waters, i.e., bidistilled water and water from a Super-Q system. Problems involved in analysing such highpurity waters are discussed.

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Analyse von Reinstwasser: Direkte Bestimmung von Ultraspuren anorganischer Verunreinigungen durch Neutronenaktivierungsanalyse mit Monostandard

     

Neue Methode zur enzymatischen Bestimmung von ?thanol in Lebensmitteln

Zusammenfassung Es wird eine neue enzymatische Methode zur Bestimmung von Äthanol in Lebensmitteln beschrieben. Äthanol wird in Gegenwart des Enzyms Alkohol-Dehydrogenase (aus Hefe, ADH, EC 1.1.1.1) durch NAD zu Acetaldehyd oxidiert. Die für diese Reaktion ungünstige Gleichgewichtslage wird durch Abfangen des Acetaldehyds mit Aldehyd-Dehydrogenase (aus Hefe, Al-DH, EC 1.2.1.5) verschoben. Die Versuchsbedingungen wurden optimiert. Die Reaktionszeit beträgt nur mehr wenige Minuten. Die Methode ist allgemein anwendbar. Besonders vorteilhaft ist auf diese Weise die Äthanolbestimmung in alkoholarmen Lebensmitteln wegen der großen Empfindlichkeit der Reaktion auszuführen. Der Test ist weitgehend störungsunabhängig und erlaubt Messungen von hoher Präzision.

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New method for the enzymatic determination of ethanol in foodstuffs

In the presence of the enzyme alcohol dehydrogenase (from yeast, ADH, EC 1.1.1.1), ethanol is oxidized to acetaldehyde by nicotinamide-adenine-dinucleotide (NAD). The unfavourable equilibrium of this reaction is displaced to the side of the reaction products by removing acetaldehyde in another enzymatic reaction, using aldehyde dehydrogenase (from yeast, Al-DH, EC 1.2.1.5). The assay conditions were optimized. The reaction time is shortened to a few minutes. In this way, ethanol can be determined in various foodstuffs, with particular advantage if the alcohol content is low. This is due to the high sensitivity of the reaction. The assay is largely free from disturbances and yields measurements of high precision.

     

Spektrophotometrische Bestimmung von Phospholipase A

Zusammenfassung Es wird eine einfach durchführbare, photometrische Bestimmung von PL-A beschrieben. Diese Methode beruht auf der Messung der bei der enzymatischen Reaktion entstehenden H⁺-Ionen anhand der Extinktionsänderung des pH-Indicators Kresolrot. Ferner werden die für dieses Testprinzip gefundenen Optimalbedingungen (Konzentration an Substrat, Ca²⁺, Einfluß verschiedener Reagentien auf die Enzymaktivität) angegeben.Es wird die Anwendbarkeit des Testprinzips auf die Messung anderer Enzyme diskutiert.

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Spectrophotometric determination of phospholipase A

A simple photometric assay for PL-A is described. This method is based on the estimation of the H⁺ ions formed during the enzymatic reaction by the change of optical density of the pH-indicator cresol red. The optimum conditions (concentration of substrate and Ca²⁺ as well as the influence of several chemicals on the activity of the enzyme) are given. Further, the application of this assay system on the estimation of other enzymes is discussed.

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Wir danken Frau U. Haid und Frl. H. Schebesch für die Mithilfe bei den experimentellen Arbeiten.     

A sensitive radiochemical assay for Coenzyme A

Summary A simple and sensitive assay for Coenzyme A (CoA) is described. The method is based on coupling the enzymatic reactions of acetyl-CoA synthetase and acetyl-CoA carboxylase. CoA is converted into acetyl-CoA with acetyl-CoA synthetase in the presence of excess ATP and acetate. Acetyl-CoA is subsequently converted into malonyl-CoA with acetyl-CoA carboxylase in the presence of excess ATP and KH¹⁴CO₃. The formation of labelled acid-stable material (i.e. malonyl-CoA) is determined. Under conditions of the assay CoA is quantitatively converted into malonyl-CoA. This procedure permits the detection of as little as 15 pmoles of CoA in biological samples. Elimination of acetyl-CoA synthetase from the reaction mixture allows for the determination of acetyl-CoA.

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Empfindlicher radiochemischer Assay für Coenzym A

Zusammenfassung Ein einfacher und empfindlicher Assay für Coenzym A (CoA) wird beschrieben, der auf einer Kopplung der enzymatischen Reaktionen von Acetyl-CoA-synthetase und Acetyl-CoA-carboxylase beruht. CoA wird mit Hilfe von Acetyl-CoA-synthetase in Gegenwart von überschüssigem ATP und Acetat zu Acetyl-CoA umgesetzt. Dieses wird anschließend mit Acetyl-CoA-carboxylase in Gegenwart von überschüssigem ATP und KH¹⁴CO₃ zu Malonyl-CoA umgewandelt. Die Bildung von markierter säurestabiler Substanz (d.h. Malonyl-CoA) wird bestimmt. Unter den Versuchsbedingungen ist die Umsetzung von CoA zu Malonyl-CoA quantitativ. Noch 15 pMol CoA können in biologischem Material erfaßt werden. Durch Entfernung von Acetyl-CoA-synthetase aus dem Reaktionsgemisch kann auch Acetyl-CoA bestimmt werden.

     

Chlorpromazine metabolism in humans. Part I

Summary A direct scan technique employing quantitative thin-layer chromatography for color samples of chlorpromazine and its metabolites by spectro-photometric method with the use of double-beam spectrodensitometer is described. Chlorpromazine and 17 metabolites have been analyzed qualitatively and quantitatively. This method can be applied for the assay of phenothiazines in both human plasma and urine. Extraction procedures for chlorpromazine and its metabolites in blood plasma and urine are presented. Advantages and disadvantages of this technique are discussed.

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Zusammenfassung Ein direktes Abtastverfahren für quantitative Dünnschichtchromatogramme von Chlorpromazin und seinen Metaboliten mit Hilfe eines Doppelstrahl-Spektrodensitometers wurde beschrieben. Chlorpromazin und 17 seiner Metaboliten wurden qualitativ und quantitativ analysiert. Das Verfahren eignet sich zur Phenothiazinbestimmung in Plasma und Harn. Extraktionsmethoden wurden angegeben. Vor- und Nachteile des beschriebenen Verfahrens wurden besprochen.

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This work was supported by USPHS Grant MH 17968.     

Kinetic- and catalytic-spectrophotometric determination of iron by pyrogallolphthalein and potassium perdisulphate in non-ionic surfactant micellar medium

Summary A simple and highly sensitive spectrophotometric determination of iron with pyrogallolphthalein in a micellar medium of non-ionic surfactant is proposed. This method is based on the kinetic-catalytic action of iron(III) upon the decomposition reaction between pyrogallolphthalein and potassium perdisulphate at pH 2.4–2.9 in the presence of Triton X 100 as a non-ionic surfactant. The calibration graph is linear over the range 10–600 ng iron(III) per 10 ml in spectrophotometry (apparent molar absorption coefficient for decomposition was 1.25×10^–6 l per mole per cm at 385 nm). The method was applied to assay and recovery tests for artificial waste water with satisfactory results (recovery 97.6%–103.5%).

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Kinetische und katalytisch-spektralphotometrische Eisenbestimmung mit Hilfe von Pyrogallolphthalein und Kaliumperdisulfat in Gegenwart eines nichtionischen Tensids

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Application of xanthene derivatives in analytical chemistry. Part LXXV. Part LXXIV see ref. [1]