Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks (original) (raw)
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Detection of C-reactive protein in a portable microfluidic immunosensor from whole human blood
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Biomedical Microdevices, 2020
Early detection and treatment of cardiovascular disease by identifying markers is important to improve the health of our citizens and simultaneously reduce the cost of health care. Therefore, there is an urgent need for early detection and treatment of asymptomatic cardiovascular disease. Herein, we have developed highly sensitive vertical flow immunokit (VFIK) for the detection of C-reactive protein (CRP), comprising an antibody/citrate conjugated gold nanoparticle in a fixed orientation surface. Our CRP test kit has been optimized with various parameters such as buffer composition, the quantity of captured antibody and sensitivity of the assay. Prominently, our developed CRP test kit is highly sensitive and rapid point of care test (POCT) which is capable to detect CRP with minimal volume (50-60 μl) and time (1-2 min) with diminutive detection up to 10 ng/ml. The reliability of our kit was evaluated and validated with spiked and clinical whole blood samples. The results demonstrated that our developed vertical flow-based point-of-care Immunokit may serve fruitful and semi-quantitative routine diagnostic for the early detection of cardiac disease.
Analytical Biochemistry, 2015
Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/ll in serum samples and 126 pg/ll in matrix-free samples. The overall sensitivity (LOQ = 75 ng/ll, R 2 = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis.
Detection of C-Reactive Protein Utilizing Magnetic Permeability Detection Based Immunoassays
Analytical Chemistry, 2005
A new sensing technology platform integrating magnetic permeability detection and a two-site heterogeneous immunoassay in a one-step analysis is described. As a platform model, measurements of C-reactive protein (CRP), a cardiac and inflammation marker, were performed in a rapid (11.5 min) high-sensitivity (hs) procedure with a low detection limit (0.2 mg/L) and accuracy (CV) 11%). The two-site heterogeneous immunoassay was performed in 1.2-mL disposable reagents vials containing solid phase (polyclonal anti-CRP conjugated silica microparticles), labeling agent (monoclonal anti-CRP conjugated superparamagnetic nanoparticles), and reaction buffer. Whole blood (20 µL) was assayed by introducing the sample into a reagent vial using a glass capillary and mixing its contents by hand for 30 s. After a 11-min sedimentation step, the vial was placed into the coil of the magnetic permeability detector, which measured the enrichment of superparamagnetic nanoparticles in the solid-phase sediment. Magnetic permeability detection and quantification is based on the principle that when paramagnetic materials are placed inside a coil, the inductance of the coil is influenced. Screening of CRP on whole blood patient samples showed good correlation with central hospital measurements for hsCRP (y) 1.018x-0.021, R 2) 0.980, n) 103) and normal range CRP (y) 1.02x + 2.53, R 2) 0.991, n) 33) analyses. The mean differences of the two methods according to the Bland and Altman plots were-0.03 (1.12 mg/L for hsCRP analysis and-3.4 (8.64 mg/L for normal range CRP analysis.
Site-directed immobilisation of antibody fragments for detection of C-reactive protein
Biosensors and Bioelectronics, 2006
C-reactive protein, CRP antibody Fab-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1 M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab) 2-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab-fragment concentration of 60 g/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 g/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 g/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.
Current analytical strategies for C-reactive protein quantification in blood
The measurement of serum C-reactive protein (CRP) levels has been given particular interest as a marker of inflammation associated with cardiovascular diseases. CRP belongs to the pentraxin family of proteins and the routine clinical analysis of CRP in blood samples is used as an important factor in primary prevention programmes together with causative and predisposing factors. This review focuses on the most representative methodologies and strategies for CRP detection and quantification that have been recently proposed, as well as reviewing those that are currently being developed for the specific, sensitive, inexpensive and high-throughput blood analysis of this protein.
Biosensors and Bioelectronics, 2014
Miniaturized diagnostic devices hold the promise of accelerate the specific and sensitive detection of various biomarkers, which can translate into many areas of medicinefrom cheaper clinical trials, to early diagnosis and treatment of complex diseases. Therefore, we report on a disposable integrated chip-based capillary immunoassay featuring a microfluidic ELISA format combining electrochemical detection and low-cost fabrication employing a dry film photoresist, Vacrel s 8100. The readily accessible carboxylate groups on the material surface allow fast and high yield immobilization of biomolecules using amine-specific coupling via reactive esters requiring no laborious surface pretreatment. The integrated microfluidic system provides a convenient platform for a flow-through immunoassay. Capillary force is used for easy reagent delivery and loading the chip channel. We performed rapid quantification of serum level of substance P, a potential biomarker of acute neuroinflammation, using the developed microfluidic immunochip. Our miniaturized assay demonstrated a sensitive electrochemical detection of the antigen at 15.4 pg ml À 1 (11.5 pM) using only 5 ml of the biological fluid while cutting the total assay preparation time in half and the read-out time to 10 min. Combining microfluidics and fabrication suitable for mass production with the capability of testing clinically relevant samples creates conditions for the construction of low-cost and portable point of care diagnostic devices with minimal auxiliary electronics.
A colorimetric homogeneous immunoassay system for the C-reactive protein
The Analyst, 2013
The C-reactive protein (CRP), which has a five repeat pentameric structure, is known to be a marker for acute inflammation and a potential risk predictor for cardiovascular disease. A simple and rapid homogeneous assay method for the detection of CRP, based on a gold nanoparticle (AuNP) aggregation induced colorimetric response, has been developed. In the technique, aggregation of CRP antibody-conjugated AuNPs is induced by addition of CRP as a consequence of its unique pentameric structure. CRP-promoted aggregation of the antibody-conjugated AuNPs results in a change of the wavelength maximum in the UV/Vis-spectrum. This homogeneous assay displays a typical hook effect, in which the signal level is directly proportional to CRP concentration until a critical concentration of CRP (the hook point) is reached. Above this concentration, the signal level decreases as the CRP concentration increases. The maximum shift in the absorption maximum was found to occur when the CRP antigen concentration is 100 ng mL À1. In order to improve the linearity of the method, we employed a procedure, which takes advantage of a saturation phenomenon that leads to the hook effect, to increase the dynamic range of the CRP assay. Specifically, the use of CRP pre-spiked serum promotes maximum aggregation at the low CRP concentrations and, as a result, leads to an increase in the dynamic range for CRP detection. The applicability of the new homogenous assay system was demonstrated by its utilization for qualitative analysis of CRP in serum samples. The combined observations made in this effort show that the method using CRP antibody-conjugated AuNPs is both rapid and simple and, consequently, it can potentially be applied to onsite diagnosis.