Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the quantification of immunoreactive trypsin.

The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561nm and emission at 585nm. The detection limits (LODs) calculated for LIF detection and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2ngml(-1), respectively. The within- and between-assay variation coefficients for the LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method, and the results were compared with those of the reference ELISA method, demonstrating a potential usefulness for the clinical assessment of IRT during the early neonatal period.

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.

Determination of trace chromium(VI) in drinking water using X-ray fluorescence spectrometry after solid-phase extraction.

A new, simple, and selective method for preconcentration and determination of Cr(VI) in aqueous samples. After adsorption in "batch mode" on Aliquat 336-AC, determinations were made directly on the solid by X-ray fluorescence spectrometry, which had the advantage of not requiring the step of elution of the chromium retained. The enrichment factor was calculated considering that the tablets obtained from 10 mL solution of Cr(VI) (1000 µg L(-1)) had a final thickness of 0.64 mm and a diameter of 16.7 mm; the volume deposited on the pellet was 0.14 cm(3). The preconcentration factor obtained was 71-fold, which was highly satisfactory for chromium trace analysis by XRF. Finally, the method was successfully applied to the determination of Cr(VI) in drinking water samples.

Slurry sampling in serum blood for mercury determination by CV-AFS.

The heavy metal mercury (Hg) is a neurotoxin known to have a serious health impact even at relatively low concentrations. A slurry method was developed for the sensitive and precise determination of mercury in human serum blood samples by cold vapor generation coupled to atomic fluorescence spectrometry (CV-AFS). All variables related to the slurry formation were studied. The optimal hydrochloric concentration and tin(II) chloride concentration for CV generation were evaluated. Calibration within the range 0.1-10 microg L(-1) Hg was performed with the standard addition method, and compared with an external calibration. Additionally, the reliability of the results obtained was evaluated by analyzing mercury in the same samples, but submitted to microwave-assisted digestion method. The limit of detection was calculated as 25 ng L(-1) and the relative standard deviation was 3.9% at levels around of 0.4 microg L(-1)Hg.

Cloud point extraction of mercury with PONPE 7.5 prior to its determination in biological samples by ETAAS.

Cloud point extraction (CPE) has been used for the pre-concentration of mercury, after the formation of a complex with 2-(5-bromo-2-pyridylazo)-5-(diethylamino)-phenol (5-Br-PADAP), and later analysis by electrothermal atomic absorption spectrometry (ETAAS) using polyethyleneglycolmono-p-nonyphenylether (PONPE 7.5) as surfactant. The chemical variables affecting the separation step were optimized. Under the optimum conditions, i.e, pH 8.5, cloud point temperature 80 degrees C, 5-Br-PADAP=4x10(-5) mol L(-1), PONPE 7.5=0.2%, sample volume=1.0 mL, an enhancement factor of 22-fold was reached. The lower limit of detection (LOD) obtained under the optimal conditions was 0.01 microg L(-1). The precision for 10 replicate determinations at 2.0 microg L(-1) Hg was 4.0% relative standard deviation (R.S.D.). The calibration graph using the pre-concentration system for mercury was linear with a correlation coefficient of 0.9994 at levels near the detection limits up to at least 16 microg L(-1). The method was successfully applied to the determination of mercury in biological samples and in certified reference material (QC METAL LL3).

Continuous-flow/stopped-flow system using an immunobiosensor for quantification of human serum IgG antibodies to Helicobacter pylori.

Conventional methods, such as gastric biopsy, enzyme-linked immunosorbent assay (ELISA), culture, require a long time for the determination of Helicobacter pylori infections. This study reports an amperometric immunoreactor for rapid and sensitive quantification of human serum immunoglobulin G (IgG) antibodies to H. pylori. Antibodies in the serum sample are allowed to react immunologically with the purified H. pylori antigens that are immobilized on a rotating disk. The bound antibodies are quantified by horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to human IgG. HRP in the presence of hydrogen peroxide catalyzes the oxidation of hydroquinone to p-benzoquinone. The electrochemical reduction back to hydroquinone is detected on a glassy carbon electrode surface at -0.15 V. The electrochemical detection can be done within 1 min, and the analysis time does not exceed 30 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.6 and 1.9 U ml-1, respectively. The amperometric immunoreactors showed higher sensitivity and lower time consumed than did the standard spectrophotometric detection ELISA method. It can also be used for rapid analysis in conventional and field conditions in biological, physiological, and analytical practices.