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Analytical Biochemistry

Sónia Ruivo, Ana M Azevedo, Duarte M F Prazeres
A microfluidic paper-based analytical device (μPADs) immunoassay for detection of the blood native biomarker D-dimer is reported. The μPAD is created by wax printing on a single piece of chromatographic paper and combined with an anti-D-dimer capture antibody and conjugates of anti-D-dimer antibody with 40 nm gold nanoparticles. The presence of D-dimer in buffer/simulated plasma samples is successfully reported for concentrations as low as 15 ng D-dimer/mL. Linearity between signal intensity and D-dimer concentration is observed up to 100 ng/mL...
September 15, 2017: Analytical Biochemistry
Zhi Li, Bingchen Li, Yunlei Zhou, Huanshun Yin, Jun Wang, Shiyun Ai
It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density...
September 15, 2017: Analytical Biochemistry
Isis C Prado, André L A Souza, David W Provance-Jr, Ricardo J Cassella, Salvatore G De-Simone
Antivenom allergy disease mediated by patient IgE is an important public health care concern. To improve detection of hypersensitive individuals prior to passive antibody therapy, an amperometric immunosensor was developed to detect reactive human IgE. Whole horse IgG3 (hoIgG3) was immobilized onto the surface of carbon or gold screen-printed electrodes through a cross-linking solution of glutaraldehyde on a chitosan film. Sera from persons with a known allergic response to hoIgG3 or non-allergic individuals was applied to the sensor...
September 14, 2017: Analytical Biochemistry
Shajie Luo, Yaqin Liu, Hanbing Rao, Yanying Wang, Xianxiang Wang
In this paper, multifunction nanoparticles (MNPs), Fe3O4@SiO2@Au MNPs, with properties of superparamagnetism, fluorescence and peroxidase-like catalytic activity were synthesized in the aqueous phase. The synthesized composites were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), powder diffraction (XRD), Fourier translation infrared spectrum (FT-IR) and fluorometer. The results show that the multifunctional nanomaterials have good magnetic and fluorescence properties...
September 13, 2017: Analytical Biochemistry
Aslı Gündoğdu, Elif Burcu Aydın, Mustafa Kemal Sezgintürk
A new, low-cost electrochemical immunosensor was developed for rapid detection of Melanoma-associated antigen 1 (MAGE-1), a cancer biomarker. The fabrication procedure of immunosensor was based on the covalent immobilization of anti-MAGE-1, biorecognition molecule, on ITO electrode by carboxyethylsilanetriol (CTES) monolayer. The biosensing MAGE-1 antigen was monitored by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) technique. Apart from these techniques, single frequency impedance (SFI) was used for investigation of antibody-antigen interactions...
September 12, 2017: Analytical Biochemistry
Atefeh Javani, Fatemeh Javadi-Zarnaghi, Mohammad Javad Rasaee
Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein...
September 11, 2017: Analytical Biochemistry
Meltem Ercan, Veli C Ozalp, Bilge G Tuna
The development of simple, reliable, and rapid approaches for molecular detection of common mutations is important for prevention and early diagnosis of genetic diseases, including Thalessemia. Oligonucleotide-gated mesoporous nanoparticles-based analysis is a new platform for mutation detection that has the advantages of sensitivity, rapidity, accuracy, and convenience. A specific mutation in β-thalassemia, one of the most prevalent inherited diseases in several countries, was used as model disease in this study...
September 8, 2017: Analytical Biochemistry
Hussein Kanso, Nicolas Inguimbert, Georges Istamboulie, Lise Barthelmebs, Carole Calas-Blanchard, Thierry Noguer
New chemiluminescence-based immunoassays for sensitive detection of 17-β estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of biotinylated estrogen derivatives. Estrogen derivatives bearing a carboxylic group (E2-COOH and EE2-COOH) on C-3 position were synthesized, covalently bound to aminated biotin and subsequently immobilized on avidin-coated microtiter plates. The assay principle was based on competition between free and immobilized estrogens for their binding to primary antibodies, with subsequent revelation using horseradish peroxidase (HRP)-labeled secondary antibodies...
September 7, 2017: Analytical Biochemistry
Valentin Muras, Björn Claussen, Hamid Nasiri, Günter Fritz, Julia Steuber
We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na(+) -translocating NADH quinone oxidoreductase (NQR) in the 96-well plate format. The assay is based on the spectrophotometric detection of NADH consumption and quinol formation. We validated the new method with known inhibitors of the NQR and optimized conditions for high-throughput screening as demonstrated by excellent Z-factors well above the accepted threshold (≥0.5). Overall, the method allows the screening and identification of potential inhibitors of the NQR, and rapid characterization of NQR variants obtained by site-specific mutagenesis...
September 7, 2017: Analytical Biochemistry
Natalie Homer, Sanjay Kothiya, Alison Rutter, Brian R Walker, Ruth Andrew
Gas chromatography mass spectrometry has been the lynchpin of clinical assessment of steroid profiles for ∼3 decades. The improvements in assay performance offered by tandem mass spectrometry were assessed. Across the spectrum of glucocorticoid and androgen analytes tested, limits of detection and quantitation were ∼20 fold lower with triple than single quadrupole systems, but the more noticeable improvement was that signal to noise was substantially improved and the linear range wider. These benefits allowed more reliable and concomitant measurement of steroids with substantially different abundances and in smaller volumes of urine...
September 5, 2017: Analytical Biochemistry
Péter Judák, Peter Van Eenoo, Koen Deventer
The tendency of peptides to adsorb to surfaces can raise a concern in variety of analytical fields where the qualitative/quantitative measurement of low concentration analytes (ng/mL-pg/mL) is required. To demonstrate the importance of using the optimal glassware/plasticware, four doping relevant model peptides (GHRP 5, TB-500, Insulin Lispro, Synachten) were chosen and their recovery from various surfaces were evaluated. Our experiments showed that choosing expensive consumables with low-bind characteristics is not beneficial in all cases...
September 5, 2017: Analytical Biochemistry
Youngjun Kim, Hyunhee Seo, Miseon Jeong, Kiheon Lee, Inho Lee, Kyeonga So, Mikyung Kim, Yookyung Lee, Seonah Kim, Taejin Kim
Cyanine 5 (Cy5) is an established fluorescent dye in microarray analysis. It is degraded rapidly when exposed to atmospheric ozone during post-hybridization washes, which leads to loss of fluorescent intensity. To minimize this undesirable effect, we coated microarray slides with sodium dodecyl sulfate (SDS) solution at post-hybridization washes. The fluorescent intensities on coated slides were more stable than those on uncoated slide. We also performed the microarrays with SDS solution for a year to check the solution's effectiveness along with seasonal changes of atmospheric ozone level...
September 4, 2017: Analytical Biochemistry
Yang Han, Shao-Yang Hou, Shang-Zhi Ji, Juan Cheng, Meng-Yue Zhang, Li-Juan He, Xiang-Zhong Ye, Yi-Min Li, Yi-Xuan Zhang
A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection...
September 4, 2017: Analytical Biochemistry
Parveen Kumar, Ranjana Jaiwal, C S Pundir
An improved amperometric biosensor for detection of creatinine was developed based on immobilization of nanoparticles (NPs) of creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) onto glassy carbon (GC) electrode. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterization of enzyme nanoparticles (ENPs). The GC electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) at different stages of its amendment...
September 1, 2017: Analytical Biochemistry
Ling Xu, Laura E Packer, Chao Li, Kojo Abdul-Hadi, Petter Veiby
The current industry practice for antibody-drug conjugate (ADC) bioanalysis includes quantification of total antibody and antibody-conjugated drug. Here, we report a novel 2-in-1 approach for measuring total antibody and protease-cleavable conjugated drug Monomethyl Auristatin E (MMAE) concurrently. This allows for the determination of the DAR (Drug Antibody Ratio) for in vivo samples, with a 3-orders linear range based on total antibody concentration from 0.1 to 100 μg/mL. Our generic, concurrent method has been cross-validated with the previously established methods in an animal study...
September 1, 2017: Analytical Biochemistry
Jefferson J Soares, Mayara B Gonçalves, Mateus C Gayer, Matheus C Bianchini, Aline C Caurio, Susana J Soares, Robson L Puntel, Rafael Roehrs, Elton L G Denardin
Fly fruit Drosophila melanogaster (DM) has been extensively employed as an in vivo model system to study pesticides toxicity. Pesticide administration to the fly traditionally involves feeding in an agar-gelled feed fly's medium (AM). However, AM method has several limitations such as uncertainty regarding the bioavailability and amount of pesticides ingested. And also high manipulation of the treated flies. We developed a new method of exposure the flies to pesticides, called Continuous Liquid Feeding (CLF)...
September 1, 2017: Analytical Biochemistry
Marcello Manfredi, Jessica Brandi, Eleonora Conte, Paolo Pidutti, Fabio Gosetti, Elisa Robotti, Emilio Marengo, Daniela Cecconi
We conducted a proteomics study in order to detect the proteomic method which provides the most complete characterization of the proteins of rice milk. In particular, we compared the results obtained from LC-MS/MS after protein precipitation with acetone or TCA, as well as the results obtained from LC-MS/MS after protein prefractionation based on SDS-PAGE (GeLC-MS/MS) or ProteoMiner™ technology (ProteoMiner-LC-MS/MS), and after peptide prefractionation based on IEF (pIEF-LC-MS/MS). A total of 158 protein species have been detect in rice milk...
August 30, 2017: Analytical Biochemistry
Yohei Sakaguchi, Tomoya Kinumi, Akiko Takatsu
An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ∼23-(A5-17 peptide) to 49-fold(B14-21 peptide), respectively, as compared with results observed in the absence of derivatization...
August 30, 2017: Analytical Biochemistry
Jeffrey E Plowman, Joy L Woods, Bede van Schaijik, Duane P Harland
A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs...
August 28, 2017: Analytical Biochemistry
Li-Ping Yang, Xiao Yan, Wen-Jia Zheng, Jun-Yi Hu, Yu-Ru Zhang, Chao-Bin Qin, Xiao-Lin Meng, Rong-Hua Lu, Fang Chen, Di-Zhi Xie, Guo-Xing Nie
Epithelial brush-border membrane vesicles (BBMVs) were isolated from the intestine of common carp and studied systematically by enzyme activity, transmission electron microscopy and immunoblotting. The uptake time course and the substrate concentration effect were assessed, and then, the ability of phlorizin and cytochalasin B to inhibit uptake was analyzed. The results show that sucrase, alkaline phosphatase and Na(+)-K(+)-ATPase activities in these vesicles were enriched 7.94-, 6.74- and 0.42-fold, respectively, indicating a relatively pure preparation of apical membrane with little basolateral contamination...
August 26, 2017: Analytical Biochemistry
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