Unprocessed Human Plasma Research Articles

Ultrasensitive immunoassay of tumor protein CA 15.3 in MCF-7 breast cancer cell lysates and unprocessed human plasma using gold nanoparticles doped on the structure of mesoporous silica

Breast cancer is the most common threat in women worldwide. Increasing death rate of diagnosed cases is the main leading cause of designing specific immunoassay for tumor marker in breast cancer. Cancer antigen 15-3 (CA 15-3) is a tumor protein for many types of cancer, most notably breast cancer. Herein, we report a sandwich-type electrochemical immunosensor based on signal amplification strategy of multiple nanocomposites to detection of CA 15-3 biomarker. The proposed immunosensor fabricated by reduced graphene oxide (rGO), poly-dopamine (PDA) and amino functionalized mesoporous silica (MCM-41-NH2) doped by gold nanoparticles composite on the glassy carbon electrode with a large surface area which was prepared a new platform to immobilization of primary antibodies and provide excellent conductivity. To further amplify the electrochemical signal, the trace tag on the foundation of gold nanoparticles (AuNPs) is coated with MCM-41-NH2 nanocomposite through thionine linking, which provides more amino groups to capture more horseradish peroxidase-labeled antibodies (HPR-Ab2) and enhances the conductivity. Under optimal conditions, the developed immunosensor exhibits excellent analytical performance for the determination of CA 15-3 with a wide linear range from 0.002 to 125 U/mL and a low limit of quantification of 0.002 U/mL. Furthermore, satisfactory results are obtained for the determination of CA 15-3 in human plasma samples, indicating the potential of the immunoassay to be applied in clinical analysis.

Electrochemical recognition of taurine biomarker in unprocessed human plasma samples using silver nanoparticlebased nanocomposite: A new platform for early stage diagnosis of neurodegenerative diseases of the nervous system.

The rapid and accurate determination of the level of taurine biomarker in various tissues and body fluids can be of great interest in the early diagnosis of several important pathologies and diseases. In the present study, an innovative electrochemical interface for quantitation of taurine based on ternary signal amplification strategy was fabricated. In this work, silver nanoparticles were electrodeposited onto green and biocompatible nanocomposite containing α-cyclodextrin as conductive matrix. Therefore, a double layer film based on α-cyclodextrin and silver nanoparticles was exploited to develop a highly sensitive electrochemical sensor for detection of taurine. Fully electrochemical methodology was used to prepare a transducer on a glassy carbon electrode which provided a high surface area towards sensitive detection of taurine biomarker. The surface morphology of electrode surface was characterized by high-resolution field emission scanning electron microscope (FE-SEM). The proposed sensing platform provides a simple tool for taurine detection. The calibration curve for taurine concentration was linear in 0.7nM to 0.1mM with low limit of quantification of 0.7nM. The practical analytical utility of the modified electrode was illustrated by determination of taurine in unprocessed human plasma samples with recovery of 90.8% to 104%.

Sensitive monitoring of taurine biomarker in unprocessed human plasma samples using a novel nanocomposite based on poly(aspartic acid) functionalized by graphene quantum dots.

The rapid and accurate determination of the level of taurine biomarker in various tissues and body fluids can be of great interest in the early diagnosis of several important pathologies and diseases. For the first time, this study reports on the electropolymerization of a low toxic and biocompatible nanocomposite "poly(aspartic acid)-graphene quantum dots (GQDs)" as a novel strategy for surface modification of glassy carbon electrode and preparation a new interface for measurement of taurine. Electrochemical deposition, as a well-controlled synthesis procedure, has been used for subsequently layer-by-layer preparation of GQDs nanostructures on poly(aspartic acid) using cyclic voltammetry techniques in the regime of -1.5 to 2V. The field emission scanning electron microscopy indicated immobilization of uniformly GQDs onto poly(aspartic acid) film. The modified electrode appeared as an effective electroactivity for detection of taurine biomarker using cyclic voltammetry, chronoamperometry, and differential pulse voltammetry. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between the excellent properties of poly(aspartic acid) as semiconducting polymer, GQDs as high density of edge plane sites, and subtle electronic characteristics to chemical modification. Under the optimized analysis conditions, the prepared sensor for detection of taurine showed a low limit of quantification 0.001mM. Finally, the resulting prepared sensor allow the quantification of these biomarkers directly in biological samples without need of derivatization schemes or sample pretreatment.

Immunosensing of breast cancer prognostic marker in adenocarcinoma cell lysates and unprocessed human plasma samples using gold nanostructure coated on organic substrate

Early stage diagnosis of cancer by monitoring the cancer biomarkers in human biological fluids is the excellent tool and the first step toward adopting efficient therapy, hindering metastasis and reducing mortality rate. An ultrasensitive electrochemical immunosensor for quantitation of tumor suppressor protein Carcinoma Antigen 15-3 (CA 15-3) based on ternary signal amplification strategy was fabricated. In this work, the antibody (horseradish peroxidase (HRP)-Labeled anti CA 15-3) was immobilized onto a green and biocompatible nanocomposite containing gold nano-shrub (Au NSs) electrochemically assembled onto thiolated graphene quantum dots (GQDs/CysA (Cysteamine)). Therefore, a novel multilayer film based on GQDs, CysA, and Au NSs was exploited to develop a highly sensitive immunosensor for detection of CA 15.3. Fully electrochemical methodology was used to prepare a new transducer on a gold surface which provided a high surface area to immobilize a high amount of the anti-CA 15.3. Under optimized condition the calibration curve for CA 15.3 concentration was linear in the range of 0.3–1 U/mL and 2–250 U/mL with detection limit of 0.011 U/mL. The method was applied to the assay of CA 15.3 in malignant cell line lysates of human breast adenocarcinoma cell line-MCF-7.

An innovative immunosensor for ultrasensitive detection of breast cancer specific carbohydrate (CA 15-3) in unprocessed human plasma and MCF-7 breast cancer cell lysates using gold nanospear electrochemically assembled onto thiolated graphene quantum dots

The accurate quantification of the level of breast cancer specific protein CA 15-3 in serum is crucial for cancer prognosis. This work, a novel and sensitive label-free immunoassay based on gold nanospear (Au NSs) electrochemically assembled onto thiolated graphene quantum dots (CysA/GQDs) for the detection of CA 15-3 antibodies. The CysA/Au NSs/GQDs hybrid interface provides a large surface area for the effective immobilization of CA 15-3 antigens, as well as it ascertains the bioactivity and stability of immobilized CA 15-3 antigens. Field emission scanning electron microscope (FE-SEM), and EDS photoelectron spectroscopies were used to monitor the sensor fabrication. Also, cyclic voltammetry was used to quantify the extent of Au NSs' surface coverage by CA 15-3 antigens. Square wave voltammetry (SWV) was employed to investigate the immunosensor fabrication and to monitor the binding events between CA 15–3 antigens-antibodies. Under optimized experimental conditions, the immunosensor displayed good sensitivity and specificity. The CA 15-3 were detected in a concentration as low as 0.11U/mL with a linear range from 0.16–125U/mL. The high sensitivity of the immunosensor may derive from the high loading of CA 15-3 antibodies on CysA/Au NSs/GQDs hybrid interface which increases the number of binding events. The method was successfully applied assay of the CA 15-3 in unprocessed human plasma samples. Also, proposed immunosensor was applied to the assay of CA 15-3 malignant cell line lysates (human breast adenocarcinoma cell line-MCF-7).

Non-enzymatic Determination of L-Proline Amino Acid in Unprocessed Human Plasma Sample Using Hybrid of Graphene Quantum Dots Decorated with Gold Nanoparticles and Poly Cysteine: A Novel Signal Amplification Strategy.

An innovative electrochemical interface for quantitation of L-proline (L-Pro) based on ternary amplification strategy was fabricated. In this work, gold nanoparticles prepared by soft template methodology were immobilized onto green and biocompatible nanocomposite containing poly as a conductive matrix and graphene quantum dots as the amplification element. Therefore, a novel multilayer film based on poly-L-cysteine, graphene quantum dots (GQDs), and gold nanoparticles (GNPs) was exploited to develop a highly sensitive electrochemical sensor for the detection of L-Pro. Fully electrochemical methodology was used to prepare a new transducer on a glassy carbon electrode, which provided a high surface area towards sensitive detection of L-Pro. The prepared electrode was employed for the detection of L-Pro. Under optimized conditions, the calibration curve for L-Pro concentration was linear in 0.5 nM - 10 mM with a low limit of quantification of 0.1 nM. The practical analytical utility of the modified electrode was illustrated by determination of L-Pro in unprocessed human plasma samples.

Aptamer based assay of plated-derived grow factor in unprocessed human plasma sample and MCF-7 breast cancer cell lysates using gold nanoparticle supported α-cyclodextrin

Platelet-derived growth factor (PDGF), a protein biomarker, is directly involved in many cell transformation processes, such as tumor growth and progression. Elevation platelet-derived growth factor (PDGF-BB) concentration in plasma could indicate the accelerating growth of metastatic breast tumors and angiogenesis. The development of an apta-assay for detection of PDGF-BB in is presented in this work. A highly specific DNA-aptamer, selected to PDGF-BB was immobilized onto a gold nanoparticles supported α-cyclodextrin and electrochemical measurements were performed in a solution containing the phosphate buffer solution with physiological pH. Variety of shapes of gold nanostructures with different sizes from zero-dimensional nanoparticles to spherical structures were prepared by one-step template (α-cyclodextrin)-assistant green electrodeposition method. Fully electrochemical methodology was used to prepare a new transducer on a gold surface which provided a high surface area to immobilize a high amount of the aptamer. The surface morphology of electrode was characterized by high-resolution field emission scanning electron microscope (FE-SEM) and energy dispersive spectroscopy (EDX). The prepared aptasensors represented different electrochemical activities toward the redox processes of PDGF-BB attributing to the size and shape of the gold nanoparticles. The aptasensor was employed for the detection of PDGF using square wave voltammetry (SWV) and Cyclic voltammetry (CV) techniques. Under optimized condition the calibration curve for PDGF-BB was linear in 0.52–1.52nM with low limit of quantification of 0.52nM. Also, under the optimized experimental conditions, the proposed aptasensor of GNPs-cubic-α-CD-Apt-Au electrode exhibited excellent analytical performance for MCF-7 cells determination, ranging from 328 TO 593 cells mL−1 with low limit of quantification of 328 cells mL−1. As a result, the electrochemical aptasensor was able to detect cancer-related targets in unprocessed human plasma samples.

Ultrasensitive electrochemical immunosensing of tumor suppressor protein p53 in unprocessed human plasma and cell lysates using a novel nanocomposite based on poly-cysteine/graphene quantum dots/gold nanoparticle

An ultrasensitive electrochemical immunosensor for quantitation of tumor suppressor protein p53 based on ternary signal amplification strategy was fabricated. In this work, p53-antibody was immobilized onto a green and biocompatible nanocomposite containing poly l-cysteine (P-Cys) as conductive matrix and graphene quantum dots (GQDs)/gold nanoparticles (GNPs) as dual amplification elements. Therefore, a novel multilayer film based on P-Cys, GQDs, and GNPs was exploited to develop a highly sensitive immunosensor for detection of p53. Fully electrochemical methodology was used to prepare a new transducer on a gold surface which provided a high surface area to immobilize a high amount of the anti-p53. Under optimized condition the calibration curve for p53 concentration was linear up to 0.000197-0.016 pM (by SWV technique) and 0.195–50 pM (by DPV technique) with lower limit of quantification of 0.065 fM. Also, linear range and lower limit of quantification of p53 in unprocessed human plasma were 0.000592-1.296 pM and 0.065 fM, respectively. The method was applied to the assay of p53 in human plasma sample and normal and malignant cell line lysates such as normal cell Line from mouse C3H (L929), colon cancer cell-HCT, prostate cancer cell line PC-3, and human breast adenocarcinoma cell line-MCF7.

An innovative immunosensor for detection of tumor suppressor protein p53 in unprocessed human plasma and cancer cell lysates

An innovative mediator-free electrochemical immunosensor for quantitation of p53 tumor suppressor protein based on signal amplification strategy was fabricated. In this work, biotin conjugated p53-antibody (anti-p53) was immobilized onto a green and biocompatible nanocomposite containing poly l-cysteine (P-Cys) as conductive matrix and 3D gold nanoparticles (GNPs) as signal amplification element. Therefore, a novel nanocomposite film based on P-Cys and GNPs was exploited to develop a highly sensitive immunosensor for detection of p53 protein. Importantly, GNPs prepared by sonoelectrodeposition method which lead to compact morphology. Fully electrochemical methodology was used to prepare a new transducer on a gold surface which provided a high surface area to immobilize a high amount of the anti-p53. The surface morphology of electrode was characterized by high-resolution field emission scanning electron microscope (FE-SEM) and energy dispersive spectroscopy (EDX). The immunosensor was employed for the detection of p53 in physiological pH using square wav voltammetry and differential pulse voltammetry (DPVs) techniques. Under optimized condition the calibration curve for p53 concentration by SWV and DPV was linear in 0.0369–50pM and 0.018–2.5pM with lower limit of quantification of 48fM and 18fM, respectively. The method was successfully applied assay of the p53 in unprocessed human plasma samples. Also, the method was applied to the assay of p53 in human plasma sample and normal and malignant cell line lysates such as (L929 normal cell Line from mouse C3H (L929), colon cancer cell-HCT, prostate cancer cell line PC-3, and human breast adenocarcinoma cell line-MCF7).

\u201cTORNADO\u201d \u2013 Theranostic One-Step RNA Detector; microfluidic disc for the direct detection of microRNA-134 in plasma and cerebrospinal fluid

Diagnosis of seizure disorders such as epilepsy currently relies on clinical examination and electroencephalogram recordings and is associated with substantial mis-diagnosis. The miRNA, miR-134 (MIR134 in humans), has been found to be elevated in brain tissue after experimental status epilepticus and in human epilepsy cells and their detection in biofluids may serve as unique biomarkers. miRNAs from unprocessed human plasma and human cerebrospinal fluid samples were used in a novel electrochemical detection based on electrocatalytic platinum nanoparticles inside a centrifugal microfluidic device where the sandwich assay is formed using an event triggered release system, suitable for the rapid point-of-care detection of low abundance biomarkers of disease. The device has the advantage of controlling the rotation speed of the centrifugal device to pump nanoliter volumes of fluid at a set time and manipulate the transfer of liquids within the device. The centrifugal platform improves reaction rates and yields by proposing efficient mixing strategies to overcome diffusion-limited processes and improve mass transport rates, resulting in reduced hybridization times with a limit of detection of 1 pM target concentration. Plasma and cerebrospinal fluid samples (unprocessed) from patients with epilepsy or who experienced status epilepticus were tested and the catalytic response obtained was in range of the calibration plot. This study demonstrates a rapid and simple detection for epilepsy biomarkers in biofluid.

Generation in Human Plasma of Misfolded, Aggregation-Prone Electronegative Low Density Lipoprotein

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(−), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(−) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(−) during incubation of unprocessed human plasma at 37°C. In addition to a higher fraction of amyloidogenic LDL(−), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone.

Evaluation of Multiprotein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

A DECREASE IN 1 H NUCLEAR MAGNETIC RESONANCE SPECTROSCOPICALLY DETERMINED CITRATE IN HUMAN SEMINAL FLUID ACCOMPANIES THE DEVELOPMENT OF PROSTATE ADENOCARCINOMA

Because human prostatic fluid contributes almost 50% of the volume of seminal plasma and this fluid contains unique prostatic metabolites such as citrate, which are markedly altered during tumorigenesis, we investigated high resolution H nuclear magnetic resonance (NMR) spectroscopy of unprocessed human seminal plasma as a rapid, noninvasive diagnostic tool for prostate adenocarcinoma. Semen and prostatic massage samples from control and tumor bearing subjects were stored frozen at -20C and thawed prior to water suppressed NMR analysis. We found that freezing produced no significant alterations in the semen NMR spectra. Quantitative NMR spectroscopy was performed by first calibrating the water suppression data acquisition sequence with a series of standard samples containing known amounts of citrate within the physiological range. Well resolved citrate resonances from the seminal plasma of 3 control subjects with prostate specific antigen (PSA) less than 1 ng/ml were integrated to give concentrations of 97 to 178 mM. Semen from a 47-year-old man with benign prostatic hyperplasia and a PSA of 5.5 ng/ml contained 156 mM citrate. In contrast, seminal plasma from 2 patients with prostate cancer, including a 46-year-old man with Gleason grade 8 and PSA 45.2 ng/ml, and a 64-year-old man with grade 6 and PSA 13.0 ng/ml, revealed citrate NMR signals corresponding to a concentration of only 28 and 24 mM, respectively. Spectra from prostatic massage fluid from a normal 23-year-old volunteer showed a citrate of 483 mM, while massage fluid from a 56-year-old patient with Gleason grade 4 cancer showed a citrate of only 1.35 mM. To our knowledge this study is the first to use high resolution NMR of semen to diagnose prostate cancer. Given the known effects of adenocarcinoma on prostate metabolism, the study indicates that high resolution H NMR can be used to measure citrate in seminal fluid, potentially providing a new, rapid, noninvasive screening method.

Physiological actions of taurine

Physiological actions of taurine

Radioimmunoassay of fibrinopeptide A - immunoreactivity of fibrinogen and fibrinogen fragments.

Abstract Anti-fibrinopeptide A antisera with different immunochemical specificities for human fibrinopeptide A (FPA), when it is part of fibrinogen or fibrinogen fragments, were used in the radioimmunoassay of FPA to demonstrate different susceptibility of native and purified molecules to degradation. Unprocessed human plasma fibrinogen had an FPA immunoreactivity with one antiserum, K2, equivalent to 0.02% of the calculated total amount of FPA in the fibrinogen, while different batches of purified fibrinogen crossreacted equivalent to 0.4–8.7% of their calculated total content of FPA in the fibrinogen. The cross-reactivity of the different batches was correlated with their dialyzable FPA immunoreactivities and was increased with storage and longer incubation times, as was also the cross-reactivity of FPA-containing fibrinogen fragments.