Architecting of a biodevice based on a screen-printed carbon electrode modified with the NiONP nanolayer and aptamer in BCM-7 detection.

In this study, an ultrasensitive and robust biodevice implemented on a screen-printed carbon electrode (SPCE) surface is introduced. The ß-casomorphin-7 (BCM-7) peptide as an early warning sign of autism disorder is distinguished in this system. The SPCE surface was directly electrodeposited with a nanolayer of the nickel oxide nanoparticles (NiONP). In next step, an Apt sequence as a capture of the BCM-7 was strongly attached on this surface. The embedded sensing interface offered some admirable characterizes to detect the BCM-7. The obtained DPV signals were reversely proportional to the concentrations of the BCM-7 through a stable binding reaction in two working linear ranges from 0.5 × 10-9-1.5 µmol l-. Also, an unrivaled limit of detection (LOD) value of 166.6 aM was achieved that is so superior by other reported methods in the BCM-7 sensing. This hand-held biodevice was satisfactorily tested for the BCM-7 detection in human urine and blood sample with an average recovery rate of ∼101.87 %. More importantly, this strategy is free from labeling steps, complex sample processing and interference from common biomolecules in blood or urine. Due to the inherent advantages of the SPCE and the NiONP, utilizing this facile sensing interface may be an ideal choice in constructing of the ultrasensitive biodevice with low cost for distinguishing of the autism disorder.

Designing of an ultrasensitive BCM-7 aptasensor based on an SPCE modified with AuNR for promising distinguishing of autism disorder.

Abstractly, in this study, an aptasensor is introduced based on a platform consisting of the gold nanorod (AuNR) on a screen printed carbon electrode (SPCE) surface. The aptasensor is applied for detection of the ß-casomorphin (BCM-7) as a promising biomarker of autism disorder. The NH2-Apt sequence is directly immobilized onto the AuNR/SPCE surface by formation of a chemisorption bond between the amine-Au groups. By incubation of the BCM-7 onto the aptasensor surface, the aptasensor directed against BCM-7 and cleverly formed a target/Apt complex to produce a measurable electrical current change. The aptasensor shows linearity over the range of 1 fmol L-1 to 25 nmol L-1 with a limit of detection (LOD) of 334 amol L-1. Furthermore, the function of the aptasensor in real samples such as human urine and plasma samples is evaluated. The achieved satisfactory results are mainly due to three main reasons including (1) the large specific surface area of the AuNR which forms a 3D network on the SPCE surface to capture more Apt sequences at the sensing interface, (2) utilizing Apt as the BCM-7 receptor with inherent unique properties to produce a synergetic effect with the AuNR, and finally, (3) effective using screen printing technology with the fantastic capability to less cost of the aptasensor preparation. There is hope that miniaturization of the proposed aptasensor may aid future efforts to detect autism symptoms as early as infancy under clinical conditions in real-world.

Influence of a Combined Gluten-Free and Casein-Free Diet on Behavior Disorders in Children and Adolescents Diagnosed with Autism Spectrum Disorder: A 12-Month Follow-Up Clinical Trial.

The use of alternative interventions, such as gluten-free and casein-free (GFCF) diets, is frequent due to limited therapies for Autism Spectrum Disorder (ASD). Our aims were to determine the influence of a GFCF diet on behavior disorders in children and adolescents diagnosed with ASD and the potential association with urinary beta-casomorphin concentrations. Thirty-seven patients were recruited for this crossover trial. Each patient consumed a normal diet (including gluten and casein) for 6 months and a GFCF diet for another 6 months. The order of the intervention (beginning with normal diet or with GFCF diet) was assigned randomly. Patients were evaluated at three time-points (at the beginning of the study, after normal diet and after GFCF diet). Questionnaires regarding behavior and autism and dietary adherence were completed and urinary beta-casomorphin concentrations were determined at each time-point. No significant behavioral changes and no association with urinary beta-casomorphin concentrations were found after GFCF diet. A 6-month GFCF diet do not induce significant changes in behavioral symptoms of autism and urinary beta-casomorphin concentrations. Further studies with a long follow-up period similar to ours and including placebo and blinding elements are needed to identify better those respondents to GFCF diets.

Aptamer-based sensing of ß-casomorphin-7.

ß-Casomorphin-7 (BCM-7), a seven amino acid peptide, is released during digestion of ß-casein A1 variant of milk which is speculated to be associated with certain diseases. Fifteen ssDNA aptamers having high affinity toward BCM-7 were identified from a 72 nt long random library after ten rounds of systematic evolution of ligands by exponential enrichment. Dissociation constant values of selected aptamers were in the range of 7.7-156.7 nM. Seq6 aptamer exhibited the lowest Kd value. Nine aptamers were evaluated for their binding toward BCM-7, BCM-9A1, and BCM-9A2 peptides, and binding was variable. SeqU5 exhibited the lowest binding with BCM-9A1 and BCM-9A2. Aptamer-coated gold nanoparticles (GNPs) resulted in color change of GNPs in the presence of BCM-7, thereby establishing recognition of BCM-7 by aptamers. The enzyme-linked aptamer-sorbent assay (ELASA) was evaluated as an assay of BCM-7 in biological fluids. BCM-7-peroxidase competed with BCM-7 in ELASA, performed with BCM-7 solution and BCM-7 spiked urine pretreated with urease, plasma, and ß-casein digest samples.

Autistic children display elevated urine levels of bovine casomorphin-7 immunoreactivity.

Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.

Autism and urinary exogenous neuropeptides: development of an on-line SPE-HPLC-tandem mass spectrometry method to test the opioid excess theory.

Autism is a complex neurodevelopmental disorder with unknown etiology. One hypothesis regarding etiology in autism is the "opioid peptide excess" theory that postulates that excessive amounts of exogenous opioid-like peptides derived from dietary proteins are detectable in urine and that these compounds may be pathophysiologically important in autism. A selective LC-MS/MS method was developed to analyze gliadinomorphin, beta-casomorphin, deltorphin 1, and deltorphin 2 in urine. The method is based on on-line SPE extraction of the neuropeptides from urine, column switching, and subsequent HPLC analysis. A limit of detection of 0.25 ng/mL was achieved for all analytes. Analyte recovery rates from urine ranged between 78% and 94%, with relative standard deviations of 0.2-6.8%. The method was used to screen 69 urine samples from children with and without autism spectrum disorders for the occurrence of neuropeptides. The target neuropeptides were not detected above the detection limit in either sample set.

Organ clearance of tyrosyl-arginine and its effect on amino acid metabolism in young sheep.

In a long-term multiple-catheter sheep model (n = 5), organ clearance of the dipeptide tyrosyl-arginine (TyrArg) and its effects on interorgan amino acid metabolism were investigated. Clearance by hindlimb and splanchnic tissues was measured during infusion into the external iliac artery and superior mesenteric artery, respectively. The hindlimb, intestine, and total splanchnic region removed 32% +/- 9.2% (mean +/- SE), 23% +/- 15%, and 33% +/- 24%, respectively, of the amount of TyrArg infused. There was a large release of tyrosine and arginine when TyrArg was infused into either the hindlimb or intestine, which was quantitatively similar to the TyrArg taken up by these organs. However, across the total splanchnic region, the baseline influx of tyrosine and arginine was not altered by infusion of TyrArg. During either clearance study, only trace amounts of TyrArg or its constituent amino acids were excreted in urine. Infusion of TyrArg produced the following effects on interorgan amino acid metabolism: (1) a reduction in the initial efflux of phenylalanine, leucine, isoleucine, and valine from the hindlimb; (2) a reduction in net efflux of citrulline by the intestine and total splanchnic tissues; and (3) a reduction in efflux of arginine and uptake of citrulline from the kidney. In conclusion, we have shown that TyrArg is cleared from the bloodstream by hindlimb (predominantly muscle) and splanchnic tissues. These results indicate that TyrArg taken up by the hindlimb and intestine was hydrolyzed to its constituent amino acids, which were released quantitatively into the circulation. Of the tissues studied, only the liver appeared to use the amino acids liberated from hydrolysis of TyrArg.(ABSTRACT TRUNCATED AT 250 WORDS)

[Maternal beta-endorphin secretion during pregnancy--measurement of immunoreactive beta-endorphin in urine].

Gestational variations in maternal beta-endorphin (beta-end) secretion were investigated by simultaneous measurements of plasma and urinary immunoreactive beta-endorphin (ir-beta-end). Urinary ir-beta-end was extracted by using a Sep-Pak C18 column and was found to be stable for at least 24 hours at room temperature. Plasma and urine (2 hour collection) samples were obtained from 41 pregnant women and 7 non-pregnant women. Almost parallel increases in plasma and urinary ir-beta-end were observed during the course of pregnancy and there was a good correlation between them. Compared with non-pregnant values, significant increases (p less than 0.001) were observed after 36 weeks of gestation. We also studied chromatographic patterns of urinary extracts of non-pregnant and pregnant women. Both of them consist of 4 peaks and resemble each other. They were essentially similar to those of plasma extracts except for the third peak which appeared between the elution position of beta-lipotropin (beta-LPH) and beta-end. In conclusion, maternal beta-end secretions increase in late gestation not only in plasma but also in urine.