Detection Of Proteins In Blood Research Articles

FEM analysis of a λ3/125 high sensitivity graphene plasmonic biosensor for low hemoglobin concentration detection.

A highly sensitive plasmonic refractive index biosensor for hemoglobin protein detection in blood is presented in the near-infrared region. The proposed Au split-ring resonator structure with an extra arm is used to increase electric field enhancement intensity in the vicinity of the nanostructure, which excites localized surface plasmon resonances in the metal-dielectric interface and leads to unity absorption. The footprint of the proposed structure is λ3/125 (λ denoting center wavelength). Through the results from the finite element method (FEM), by variation of the spacer material, and inserting a graphene layer between the spacer and the gold nanostructure, maximum sensitivities of 1804.1 nm/RIU and 2448.45 nm/RIU are achieved, respectively.

Protein detection in blood with single-molecule imaging.

The ability to characterize individual biomarker protein molecules in patient blood samples could enable diagnosis of diseases at an earlier stage, when treatment is typically more effective. Single-molecule imaging offers a promising approach to accomplish this goal. However, thus far, single-molecule imaging methods have not been translated into the clinical setting. The detection limit of these methods has been confined to the picomolar (10-12 M) range, several orders of magnitude higher than the circulating concentrations of biomarker proteins present in many diseases. Here, we describe single-molecule augmented capture (SMAC), a single-molecule imaging technique to quantify and characterize individual protein molecules of interest down to the subfemtomolar (<10-15 M) range. We demonstrate SMAC in a variety of applications with human blood samples, including the analysis of disease-associated secreted proteins, membrane proteins, and rare intracellular proteins. SMAC opens the door to the application of single-molecule imaging in noninvasive disease profiling.

Ultra-Trace Protein Detection by Integrating Lateral Flow Biosensor with Ultrasound Enrichment.

Lateral flow biosensor (LFB) is one of the most successful and applied commercial detection methods for food safety, drug abuse, and disease. Here, we integrated the ultrasound enrichment as sample preparation with LFB to achieve the ultra-trace protein detection in blood. When the ultrasound field is applied, the interaction between the acoustic field and gold nanoparticles can gather specifically modified gold nanoparticles toward pressure nodes in seconds and enrich target proteins. Such an approach can detect protein with a linear range of 1-20 ng mL-1 and detection limit of 0.58 ng mL-1 in blood within 20 min, which enormously reduces false positive readings caused by interference in real blood samples with complex components. Such a microchip that integrated acoustic enrichment with LFB shows great potential in detecting ultra-trace biomarkers for clinical diagnosis.

Multiwell Plate Impedance Analysis of a Nanowell Array Sensor for Label-Free Detection of Cytokines in Mouse Serum

We present a novel method for label-free detection of tumor necrosis factor alpha (TNF-α) in serum after immobilization by electric field. Detection of proteins in blood using label-free impedance-based techniques is difficult due to high salt concentration of the matrix, which results in charge screening. The nanowell array sensor provides enhanced electrochemical sensitivity by electric field focusing in the nanoscale volume wells. Here, an on-chip multiwell plate sensing platform was fabricated and tested. The sensor performance through testing in mouse serum at protein concentrations between 10 and 500 ng/l has been demonstrated. This detection modality is advantageous to many label-free electronic sensors in that signal power scales will increase in salt concentration, thus improving the sensitivity of the platform.

Protein Detection in Blood with Single-Molecule Imaging

The ability to accurately identify and characterize proteins in blood samples would facilitate disease diagnosis and monitoring. Current blood tests, including ultra-sensitive assays, depend on ensemble measurements which are hampered by background detection errors. Single-molecule imaging approaches, by contrast, directly inspect individual target proteins, offering much greater reliability than ensemble methods. However, the detection limit of single-molecule imaging has thus far been confined to the picomolar (10−12 M) range, and these approaches have not been previously translatable to clinical samples. Here we describe Single-Molecule Augmented Capture (SMAC), a single-molecule imaging technique to visualize and quantify individual proteins-of-interest down to the sub-femtomolar (<10−15 M) range with minimal detection errors, even in complex fluids such as blood. We demonstrate SMAC in a wide variety of applications with human blood samples, including the analysis of secreted proteins (prostate-specific antigen and anti-p53 autoantibodies), membrane proteins (programmed death-ligand 1), and rare intracellular proteins (mutant p53). SMAC opens the door to the use of single-molecule imaging in non-invasive disease profiling. This platform can be adapted to multiplex or high-throughput formats to characterize heterogeneous biochemical and structural features of circulating proteins-of-interest.

Protein detection in blood via a chimeric aptafluorescence assay: toward point-of-care diagnostic devices

Paper-based analytics allows building portable and disposable devices for point-of-care (POC) diagnosis. Conventional methods for quantifying proteins exhibit substantial disadvantages related to costs and difficulty of the technique when used in settings where fast and cost-effective assays are needed. We report the successful application of a simple, rapid, easy to use, and label-free aptasensor strategy based on the selective fluorescence of the NMM IX dye. For the probe design, the three-dimensional (3-D) structures of the DNA components were carefully analyzed using software for the 3-D visualization of crystallographic structures. The chimeric aptafluorescence molecule consists of two modules, a detection aptamer and a transduction sequence that induces the specific fluorescence of NMM IX. In the presence of thrombin, a fluorescent spot visible to the naked eye can be observed. The fluorescent response is directly proportional to protein concentration and can be easily quantified colorimetrically using a low-cost microscopy system. The recognition probe design might be adaptable to other relevant biological analytes by changing the sequence of the aptamer. This proof of principle represents a contribution to the development of useful, cheap, reliable, and simple protein quantification assays for POC testing.

Residual nanoparticle label immunosensor for wash-free C-reactive protein detection in blood

Current diagnostic immunotechnologies are universally based on the measurement of the bound label-antibody fraction in direct binding or sandwich-assay type approaches with various detection techniques (e.g. enzyme-linked immunosorbent assay or ELISA) on solid stationary phase surface. Here an alternative reciprocal approach is presented based on the detection of the non-bound fraction of nanoparticle-labelled antibodies using microparticles as solid support. The advantage of detecting the non-bound fraction of the labelled antibody instead of the bound fraction is the high dynamics and the suggested increased flexibility in the selection of the detection mode. No actual washing steps are required as the bound and non-bound fractions of the detection nanoparticle label are separated using physical separation rather than consecutive washing repeats. The quantitative proof-of-concept set-up was demonstrated through blood-based detection of C-reactive protein (CRP). A blood sample containing CRP was diluted 1/50 and measured in 15-min resulting in a linear response at a range from 1 to 30μg/ml. The lowest limit of detection was below 0.03μg/ml and the assay coefficient of variation ranged from 0.3 to 9%. The nanoparticle-based residual label detection outperformed the corresponding molecular label method providing wider applicability with nearly an order of magnitude higher signal-to-background ratio for novel assay configurations in clinical diagnostics practices.

1C34 マイクロ流路中に固定化した金ナノ粒子の局在表面プラズモン共鳴を利用した血中タンパク質の非標識検出(OS14-3:バイオMEMS(3))

1C34 マイクロ流路中に固定化した金ナノ粒子の局在表面プラズモン共鳴を利用した血中タンパク質の非標識検出(OS14-3:バイオMEMS(3))

Ultrasensitive and real-time detection of proteins in blood using a potentiometric carbon-nanotube aptasensor

Potentiometric sensing represents the preferred technique in many routine measurements of pH and ions. Unfortunately, the simplicity of the technique has not been exploited so far in high throughput biomolecular sensing. In this work, we demonstrate the capabilities of the hybrid functional material carbon nanotubes/aptamer for the creation of a new generation of nuclease-resistant aptasensors using the potentiometric transduction capabilities of single-walled carbon nanotubes in combination with the recognition capabilities of a protein-specific RNA aptamer. The aptasensor was used to detect and identify disease-related proteins at attomolar concentration values in a rapid and non-expensive way. The variable surface glycoprotein from African Trypanosomes was chosen as an ideal model system for a pathogenic exoantigen protein in a clinical sample. Variations in the electromotive force are achieved in real-time upon the direct addition of diluted real blood samples containing the target protein thus eliminating the need of preliminary matrix removal. This work would open the door to real-time diagnostic assays for a wide range of diseases, but also to the rapid molecular detection of several proteins in truly customizable protein biosensing platforms.

Abstract 4899: Development of a quantitative measurement of p53 – p53 antibody interactions

Abstract We are developing methods to quantify antibody interactions that includes a quartz crystal microbalance (QCM) system to measure, on a molecular basis, the interaction of p53 and anti-p53 antibodies. Previously, as a model system, we developed a reference device consisting of p53 protein (human wild type), characterized by mass spectroscopy and immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with immunohistochemical (IHC) assays that incorporate commercially available anti-p53 antibodies and probes. In the current study, we compared three different monoclonal antibodies (clones: BP53.12, PAb 1801 and DO-1) for their ability to detect p53 in HT1080 cells. Both visible microscopy (diaminobenzidine probe) and flow cytometry (AlexaFluor 488 probe) showed significant increases in staining intensity compared to controls without primary antibody. However, quantification of these observations requires direct measurement of the concentration of the p53 and the concentrations and specificities of the antibodies. For this the p53 protein is covalently immobilized on glass coated quartz crystals and the resonance frequency shift is followed in-situ. The functionalized sensor is then incubated with the anti-p53 antibody, which provides a direct gravimetric measure of antibody-antigen binding. This allows the comparison of the surface immobilized protein concentrations with those previously obtained by fluorescence measurement. The p53 functionalized QCM system offers an independent measure of surface immobilized protein concentration and is essential in the development of our IHC calibration prototypes. These results also provide a benchmark for comparing systems that may be used in developing molecular diagnostic assays such as aspiration biopsy analysis and the detection of biomarker proteins in blood and urine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4899. doi:10.1158/1538-7445.AM2011-4899

Endogenous retrovirus type W GAG and ENV antigenaemia in serum of schizophrenic patients

Recent and independent molecular studies have shown an association between Human Endogenous Retroviruses type ''W'' family (HERV-W) and Schizophrenia, mostly by PCR studies, but none has yet addressed specific antigen detection in living patients. HERV-W GAG (Matrix epitope) and ENV (Envelope epitope) proteins were dosed in the serum of patients with Schizophrenia and controls with a dedicated immunoassay set-up with specific monoclonal antibodies to either antigen. In schizophrenic patients, positive antigenaemia for ENV and for GAG was found in nearly 50% of cases. Difference with healthy controms was very significant (p < 0,01). All positive sera for ENV were also positive for GAG. Significant correlation was found between ''ENV'' antigenaemia, a protein causing dysimmune inflammatory effects and C-Reactive Protein levels-CRP, a systemic inflammation biomarker now known to be a hallmark of neurodegenerative forms of Schizophrenia. It is thus quite interesting to observe that, beyond association with systemic inflammation, HERV-W protein detection in blood here associates ENV and GAG antigen with coincident detection by three different monoclonal antibodies targeting distinct epitopes. This raises the question of coincident release of both proteins in the bloodstream or of HERV-W retroviral particles (such as ''Multiple Sclerosis Associated RetroViral'' particles, MSRV) as described by others in Schizophrenia as in Multiple Sclerosis. As viruses such as Influenza, long associated with risk for schizophrenia through perinatal infections have also revealed to activate HERV-W elements in human cells, we may now provide an hypothesis for a pathogenic cascade in association with HERV-W associating the following factors: environment factors long associated with early perinatal risk consisting in certain infectious agents, genetic factors represented by this retroviral endogenous family, the production of its pro-inflammatory ENV protein, and known ''inflammation-mediated'' neurotoxicity. Our present results thus confirm that HERV-W studies have opened a novel avenue of research in a neurodegenerative type of schizophrenia.

Detection of blood proteins in ancient human bone using ELISA: A comparative study of the survival of IgG and albumin

AbstractABSTRACT The immunological identification of species‐specific blood proteins in skeletal remains has an important role in the reconstruction of ancient dietary, ritual and domestic behaviour. However, which protein provides the most suitable target for such work has not been considered previously, and the present investigation was carried out on human bone to assess the relative merits of IgG and albumin.Extracts of bone from 31 individuals (from the English Civil War, medieval, Early Saxon, Roman, Iron Age and Bronze Age periods) were tested by an enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibodies. Albumin was detected in 23 of the 31 skeletons, including those from the Iron and Bronze Ages, whereas IgG was identified in only one; this difference was very highly significant (P &lt; 0.0005, χ2 30.0). The better detection rate for albumin was thought to be due to its higher original blood level, inherent differences in survival pattern being considered unlikely. Testing the same extract for both proteins in the same assay system ensured that any effects due to soil factors, burial conditions, physical integrity of the bone, chronological age, amount of original specimen, method of analysis and type of reagent were the same for each part of the study, thus permitting a valid comparison of antigen survival to be made. Control samples, including fresh and ancient animal bone extracts and human and animal sera, confirmed that the results were consistent and specific, with no cross‐reactivity between human and animal material, and that as little as 10 ng of protein was detectable.In summary, the investigation compared the suitability of IgG and albumin for osteoarchaeological studies using a highly specific, sensitive and versatile ELISA; the results showed that albumin was a far better target molecule for such work and that it can survive in ancient bone for long periods of time.