Hybrid-Integrated Biosensor for Express Determination of Protein Markers of Diseases based on Molecular Recognition and Direct Fluorimetric Detection

2021 ◽  
Vol 23 (6) ◽  
pp. 326-332
Author(s):  
N.O. Sitkov ◽  
◽  
T.M. Zimina ◽  
V.V. Luchinin ◽  
A.A. Kolobov ◽  
...  
Keyword(s):  
Molecular Recognition ◽  
Spatial Configuration ◽  
Limit Of Detection ◽  
Troponin T ◽  
Protein Structures ◽  
Microfluidic Channel ◽  
Protein Markers ◽  
Protein Marker ◽  
Aromatic Amino Acid Residue ◽  
Peptide Probes

Ways of creating new generation biosensors for multiparametric express diagnostics based on molecular recognition and direct fluorimetric registration of a peptide aptamer — protein marker complex were considered. The biosensor platform comprises a microfluidic channel for delivery sample solutions, coupled with flow-through zones containing covalently attached arrays of peptide probes — aptamers. An outer glass window of the biochip assembly contains a layer of luminophore ZnS:Cu, bound on it via an acrylic lacquer and intended for the re-emitting native fluorescence of bound proteins into the longer wavelength range, more efficient in registering signals with CMOS sensors. The aptamers were designed using "Protein 3D" program for analysis of spatial complementarity of protein structures. The peptide, complementary to Troponin T, was modified by replacement of aromatic amino acid residue while maintaining the spatial configuration. The complementarity of peptide and Troponin T was confirmed using a capillary electrophoresis-on-a-chip. Biosensors are manufactured using thick-film technology and photolithography. The fluorescence of marker proteins was excited using UV-LED with a radiation wavelength of 275 nm. The limit of detection achieved for Troponin T was 6 ng/ml.

Pressure threshold for fluid loss from the peritoneal cavity

1996 ◽  
Vol 270 (2) ◽  
pp. F377-F390 ◽  
Author(s):  
M. F. Flessner ◽  
A. Schwab
Keyword(s):  
Abdominal Wall ◽  
Peritoneal Cavity ◽  
Loss Rate ◽  
Lymph Flow ◽  
The Body ◽  
Fluid Loss ◽  
Protein Markers ◽  
Protein Marker ◽  
Fluid Movement ◽  
Pressure Threshold

Ascites or dialysis fluid in the peritoneal cavity causes fluid loss from the cavity to the body. Experiments in animals and in humans have shown that the fluid loss rate increases with large increments in the intraperitoneal hydrostatic pressure (Pip). We hypothesized that there is a low-threshold Pip above which this fluid loss occurs. Because the full Pip force is exerted across the abdominal wall (AW), we further hypothesized that fluid movement into the abdominal wall would vary directly with the Pip. To address these questions, we dialyzed rats for 3 h in the supine position at constant levels of Pip with isotonic and hypertonic dialysis solutions containing a protein marker of fluid movement. We measured total fluid loss, AW fluid-marker concentration, and lymph flow. With variation of Pip from 0 to 8 cmH2O, we found that 1) lymph flows (0.61 +/- 0.03 ml/h) were not dependent on Pip, 2) measured isotonic fluid loss rate varied from 0.29 +/- 0.06 ml/h at 0 cmH2O to 0.62 +/- 0.02 at 2 cmH2O and then rose in a linear fashion to 5.06 +/- 0.10 ml/h at 8 cmH2O, 3) fluid movement into the AW paralleled the measured fluid loss rate, and 4) protein clearance from the cavity overestimated the true fluid loss because of adsorption of the marker to the peritoneal surface. We conclude that, although peritoneal lymph flow is not dependent on intraperitoneal hydrostatic or osmotic pressure, fluid loss from the cavity and fluid loss to the abdominal wall are directly proportional to Pip > 2 cmH2O. We also note that protein markers of fluid movement require correction for tissue surface adsorption for accurate results.

scholarly journals Detecting Glucose Levels in Blood Plasma and Artificial Tear by Au(I) Complex on the Carbopol Polymer: A Microfluidic Paper-Based Method

Polymers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 1001 ◽  
Author(s):  
Jong-Jheng Luo ◽  
Sheng-Wei Pan ◽  
Jia-Hui Yang ◽  
Tian-Lin Chang ◽  
Peng-Yi Lin ◽  
...  
Keyword(s):  
Plasma Glucose ◽  
Limit Of Detection ◽  
Microfluidic Channel ◽  
Glucose Sensing ◽  
Single Type ◽  
Dual Emission ◽  
Glucose Levels ◽  
Long Term Storage ◽  
Tear Glucose ◽  
Response Region

We report on a selective paper-based method and a microfluidic paper-based analytical device (μPAD) for the detection of human plasma glucose and tear glucose using carbopol polymer-encapsulated Au(I) complex (AuC2C6H4OMe)2(Ph2P(C6H4)3PPh2), (B5). To the best of our knowledge, this demonstrates for the first time the glucose sensing based on dual emission, i.e., fluorescence and phosphorescence, of a single type molecule on the carbopol polymer. Upon addition of human blood treated with anticoagulants to μPADs, plasma is separated from the blood and flows into the response region of the μPADs to react with carbopol polymer-encapsulated B5, in which the ratiometric luminescence is analyzed. The plasma glucose concentration can be quantitively detected at 1.0–50.0 mM on paper, and tear glucose can be detected at 0.1–4.0 mM on μPADs. Owing to the structural design, this device has superior ratiometric changes of dual emission over other Au(I) complexes for signal transduction. The encapsulation of carbopol polymer also offers long-term storage stability. In tear measurement, carbopol polymer is not only used to encapsulate enzyme to remain the enzyme’s activity, but also played as a glue (or media) to connect microfluidic channel and response region. This further improves the sensitivity and limit of detection for glucose. Moreover, this sensor provides a faster response time, a wider range for glucose sensing than reported previously, and no statistical difference of the data from a commercial glucometer, allowing for practical diagnosis of diabetes and healthy individuals.

scholarly journals Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA.

1997 ◽  
Vol 8 (7) ◽  
pp. 1233-1242 ◽  
Author(s):  
J Nunnari ◽  
W F Marshall ◽  
A Straight ◽  
A Murray ◽  
J W Sedat ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescence Microscopy ◽  
Fluorescent Dyes ◽  
Mitochondrial Protein ◽  
Mitochondrial Proteins ◽  
Dimensional Structure ◽  
Wide Field ◽  
Protein Markers ◽  
Protein Marker ◽  
Mitochondrial Transmission

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.

scholarly journals Re-evaluating the conventional wisdom about binding assays

2020 ◽  
Author(s):  
Brandon D. Wilson ◽  
H. Tom Soh
Keyword(s):  
Molecular Recognition ◽  
Conceptual Understanding ◽  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Conventional Wisdom ◽  
Binding Assays ◽  
Digital Measurements ◽  
Assay Design ◽  
Recognition Systems

AbstractAnalytical technologies based on binding assays have evolved substantially since their inception nearly 60 years ago, but our conceptual understanding of molecular recognition has not kept pace. Indeed, contemporary technologies such as single-molecule and digital measurements have challenged, or even rendered obsolete, core aspects of the conventional wisdom related to binding assay design. Here, we explore the fundamental principles underlying molecular recognition systems, which we consider in terms of signals generated through concentration-dependent shifts in equilibrium. We challenge certain orthodoxies related to binding-based detection assays, including the primary importance of a low KD and the extent to which this parameter constrains dynamic range and limit of detection. Lastly, we identify key principles for designing binding assays optimally suited for a given detection application.

scholarly journals Electrochemical ELISA Protein Biosensing in Undiluted Serum Using a Polypyrrole-Based Platform

Sensors ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 2857 ◽  
Author(s):  
Sunil K. Arya ◽  
Pedro Estrela
Keyword(s):  
Serum Proteins ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Markers ◽  
Necrosis Factor Alpha ◽  
Force Microscopy ◽  
Atomic Force ◽  
Tnf Α ◽  
Undiluted Serum

An electrochemical enzyme-linked immunosorbent assay (ELISA) biosensor platform using electrochemically prepared ~11 nm thick carboxylic functionalized popypyrrole film has been developed for bio-analyte measurement in undiluted serum. Carboxyl polypyrrole (PPy-COOH) film using 3-carboxy-pyrrol monomer onto comb-shaped gold electrode microarray (Au) was prepared via cyclic voltammetry (CV). The prepared Au/PPy-COOH was then utilized for electrochemical ELISA platform development by immobilizing analyte-specific antibodies. Tumor necrosis factor-alpha (TNF-α) was selected as a model analyte and detected in undiluted serum. For enhanced performance, the use of a polymeric alkaline phosphatase tag was investigated for the electrochemical ELISA. The developed platform was characterized at each step of fabrication using CV, electrochemical impedance spectroscopy and atomic force microscopy. The bioelectrodes exhibited linearity for TNF-α in the 100 pg/mL–100 ng/mL range when measured in spiked serum, with limit of detection of 78 pg/mL. The sensor showed insignificant signal disturbance from serum proteins and other biologically important proteins. The developed platform was found to be fast and specific and can be applicable for testing and measuring various biologically important protein markers in real samples.

scholarly journals The Effects of Mechanical Stimulation on Controlling and Maintaining Marrow Stromal Cell Differentiation Into Vascular Smooth Muscle Cells

2015 ◽  
Vol 137 (2) ◽  
Author(s):  
Raphael Yao ◽  
Joyce Y. Wong
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dynamic Strain ◽  
Protein Markers ◽  
Protein Marker ◽  
Significant Step ◽  
Promising Source

For patients suffering from severe coronary heart disease (CHD), the development of a cell-based tissue engineered blood vessel (TEBV) has great potential to overcome current issues with synthetic graft materials. While marrow stromal cells (MSCs) are a promising source of vascular smooth muscle cells (VSMCs) for TEBV construction, they have been shown to differentiate into both the VSMC and osteoblast lineages under different rates of dynamic strain. Determining the permanence of strain-induced MSC differentiation into VSMCs is therefore a significant step toward successful TEBV development. In this study, initial experiments where a cyclic 10% strain was imposed on MSCs for 24 h at 0.1 Hz, 0.5 Hz, and 1 Hz determined that cells stretched at 1 Hz expressed significantly higher levels of VSMC-specific genetic and protein markers compared to samples stretched at 0.1 Hz. Conversely, samples stretched at 0.1 Hz expressed higher levels of osteoblast-specific genetic and protein markers compared to the samples stretched at 1 Hz. More importantly, sequential application of 24–48 h periods of 0.1 Hz and 1 Hz strain-induced genetic and protein marker expression levels similar to the VSMC profile seen with 1 Hz alone. This effect was observed regardless of whether the cells were first strained at 0.1 Hz followed by strain at 1 Hz, or vice versa. Our results suggest that the strain-induced VSMC phenotype is a more terminally differentiated state than the strain-induced osteoblast phenotype, and as result, VSMC obtained from strain-induced differentiation would have potential uses in TEBV construction.

scholarly journals Simultaneous quantitative profiling of clinically relevant immune markers in neonatal stool swabs to reveal inflammation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Veronika Vidova ◽  
Eliska Benesova ◽  
Jana Klanova ◽  
Vojtech Thon ◽  
Zdenek Spacil
Keyword(s):  
Total Protein ◽  
Limit Of Detection ◽  
Eosinophil Cationic Protein ◽  
Multiplex Assay ◽  
Microbial Colonization ◽  
Food Allergies ◽  
Coefficient Of Determination ◽  
Protein Markers ◽  
Immune Markers ◽  
Cationic Protein

AbstractAn aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.

scholarly journals Study of the Fabrication Technology of Hybrid Microfluidic Biochips for Label-Free Detection of Proteins

Micromachines ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 20
Author(s):  
Nikita Sitkov ◽  
Tatiana Zimina ◽  
Alexey Kolobov ◽  
Evgeny Sevostyanov ◽  
Valentina Trushlyakova ◽  
...  
Keyword(s):  
Technological Process ◽  
Biological Fluid ◽  
Protein Structures ◽  
Microfluidic System ◽  
Diagnostic Tools ◽  
Fluorometric Detection ◽  
Label Free ◽  
Protein Markers ◽  
Label Free Detection ◽  
Microfluidic Biochips

A study of the peculiarities and a comparative analysis of the technologies used for the fabrication of elements of novel hybrid microfluidic biochips for express biomedical analysis have been carried out. The biochips were designed with an incorporated microfluidic system, which enabled an accumulation of the target compounds in a biological fluid to be achieved, thus increasing the biochip system’s sensitivity and even implementing a label-free design of the detection unit. The multilevel process of manufacturing a microfluidic system of a given topology for label-free fluorometric detection of protein structures is presented. The technological process included the chemical modification of the working surface of glass substrates by silanization using (3-aminopropyl) trimethoxysilane (APTMS), formation of the microchannels, for which SU-8 technologies and a last generation dry film photoresist were studied and compared. The solid-state phosphor layers were deposited using three methods: drop application; airbrushing; and mechanical spraying onto the adhesive surface. The processes of sealing the system, installing input ports, and packaging using micro-assembly technologies are described. The technological process has been optimized and the biochip was implemented and tested. The presented system can be used to design novel high-performance diagnostic tools that implement the function of express detection of protein markers of diseases and create low-power multimodal, highly intelligent portable analytical decision-making systems in medicine.

scholarly journals Paragangliomas of the head and neck: clinical, morphological and immunohistochemical aspects

2001 ◽  
Vol 119 (3) ◽  
pp. 114-118 ◽  
Author(s):  
Pedro de Alcântara de Andrade Filho ◽  
Abrão Rapoport ◽  
Venâncio Avancini Ferreira Alves ◽  
Odilon Victor Porto Denardin ◽  
Josias de Andrade Sobrinho ◽  
...  
Keyword(s):  
Head And Neck ◽  
Therapeutic Approach ◽  
S100 Protein ◽  
Protein Markers ◽  
Protein Marker ◽  
Sustentacular Cells ◽  
Definition Of ◽  
Clinical Records ◽  
Histological Architecture ◽  
Head And Neck Paragangliomas

CONTEXT: Protein marker positivity can assist in the definition of the therapeutic approach towards head and neck paragangliomas. The establishment of the therapeutic approach should incorporate the results of such an investigation. OBJECTIVE: To establish criteria for benignancy and malignancy of vagal and jugular-tympanic paragangliomas, via the study of the relationships of sex, age, tumor size, duration of complaints, site, family history, presence of metastases, treatment, histological architecture and cell type with the immunohistochemical reactions to S100 protein, chromogranin and AgKi67. DESIGN: A retrospective study of histological and clinical records. SETTING: The Heliópolis and Oswaldo Cruz tertiary general hospitals, São Paulo. SAMPLE: 8 cases of head and neck paragangliomas. MAIN MEASUREMENTS: Determination of degree of positivity to paragangliomas via immunohistochemical reactions. RESULTS: 1). The protein markers for the principal cells (AgKi67 and chromogranin) were sensitive in 100% of the tumors when used together. 2). S100 protein was well identified in the cytoplasm and nucleus of sustentacular cells and underwent reduction in the neoplasias. CONCLUSIONS: Chromogranin was proven to be a generic marker for neuroendocrine tumors; S100 protein was positive in all 8 cases and the AgKi67 had low positivity in all cases.

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