Immunoassay For C-reactive Protein Research Articles

A rapid and facile immunoassay for C-reactive protein using PDMS-based digital magnetofluidics

BackgroundC-reactive protein has been reported as a biomarker of inflammation caused by acute injury, infection or tissue damage and also a prediction marker of cardiovascular diseases. Commonly, the gold standard for the detection of CRP is enzyme-linked immunosorbent assays (ELISAs). Normally, traditional immunoassays in multiwell plates typically suffer from prolonged assay time due to slow mass transport controlled by diffusion. Herein, a PDMS based magnetofluidic approach has been applied for a rapid and facile immunoassay using a sandwich enzyme-linked immunosorbent assay (ELISA) for the analysis of CRP. ResultsDue to the superhydrophobic PDMS, droplets of reagent and sample solutions were obtained when pipetting all solutions onto the PDMS substrate. These droplets were individually controlled by an external magnet to perform the assays. Magnetic beads immobilized with a capture antibody were not only used for immunomagnetic separation (IMS) of the captured CRP from the sample matrix, but also used as a carrier for droplet movement on the magnetofluidic device, expediting the immunoassay procedure, especially washing steps. The immunoassay of CRP was successfully performed within 1 h with a limit of detection of 0.015 mg L−1 in the concentration range of 0.1–10 mg L−1. The recovery percentages of CRP spiked in human serum were found in the range of 90–114 % with %RSD of less than 5 %, indicating acceptable accuracy and precision. SignificanceBy individually controlling the droplet movement using an external magnet, all steps of immunoassays were simply and rapidly performed. In addition, the microfluidic format allows for small volumes of reagents and samples and rapid assay kinetics. Therefore, the proposed magnetofluidic approach has shown its potential of becoming a rapid, facile and cost-effective method to perform traditional immunoassays in a variety of applications. In addition, the proposed approach is also particularly well-suited for analyses/reactions with multiple steps.

Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies.The developed method exhibited a linear detection range of approximately 6–200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%–102%. These results indicate robust reproducibility of the method.To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits.In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Signaling redox probe/DNA aptamer complexes on a new POP/2D WSe2 composite-based immunosensor towards the simultaneous detection of three-protein overexpression as an alternative severe SARS-COV-2 infection diagnosis

The coronavirus disease 2019 (COVID-19) has become a global pandemic in which patients with symptoms are infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical investigation of human immune response cytokines or/and overexpressed proteins at trace cut-off levels can be valuable to monitor body function failures and, eventually, improve the prognosis for patients. Nevertheless, the currently available technologies are still limited. Herein, we provide the first sandwich-like immunosensor that has the potential to detect multiple protein biomarkers in a single measurement. The sandwich-like structure was created by coating a three-screen printed carbon electrode (3SPCE) array with a porous organic polymer (POP)/2D WSe2 nanocomposite. This system enables the simultaneous immunoassay of C-reactive protein (CRP), native cardiac troponin I (cTnI), and procalcitonin (PCT), which together provide a panel of symptom-diagnostic biomarkers for the presence and severity of COVID-19 infection. The biomarker detection principle relies on three antibodies as bioreceptor probes bound on the modified 3SPCE and three aptamers to generate three redox probe complexes as signal amplifiers. After incorporating three redox species (Cd2+, methylene blue (MB), and Ag+) into the aptamer structure, well-differentiated oxidation peaks can be achieved, indicating the concentrations of these three proteins. Under the optimized conditions of the stepwise fabrications, the linear dynamic range of three-protein detection by our proposed multiplex biosensor ranges from 100 to 50,000 pg mL–1. The limits of detection (LODs) are as low as 62, 81, and 44 pg mL–1 for the detection of CRP, cTnI, and PCT, respectively. As proof of the concept, this multiplexed assay demonstrates outstanding analytical performance with excellent reproducibility, high sensitivity and selectivity, and acceptable recoveries of the detection in human serum. Therefore, the multiplexed sensor could be an alternative tool in the field of clinical analysis for the diagnosis of COVID-19 severity or other emerging infectious diseases.

Patient Stratification for Antibiotic Prescriptions Based on the Bound-Free Phase Detection Immunoassay of C-Reactive Protein in Serum Samples.

C-reactive protein is a well-studied host response biomarker, whose diagnostic performance depends on its accurate classification into concentration zones defined by clinical scenario-specific cutoff values. We validated a newly developed, bead-based, bound-free phase detection immunoassay (BFPD-IA) versus a commercial CE-IVD enzyme-linked immunosorbent assay (ELISA) kit and a commercial CE-IVD immunoturbidimetric assay (ITA) kit. The latter was performed on a fully automated DPC Konelab 60i clinical analyzer used in routine diagnosis. We classified 53 samples into concentration zones derived from four different sets of cutoff values that are related to antibiotic prescription scenarios in the case of respiratory tract infections. The agreements between the methods were ELISA/ITA at 87.7%, ELISA/BFPD-IA at 87.3%, and ITA/-BFPD-IA at 93.9%, reaching 98-99% in all cases when considering the calculated relative combined uncertainty of the single measurement of each sample. In a subgroup of 37 samples, which were analyzed for absolute concentration quantification, the scatter plot slopes' correlations were as follows: ELISA/ITA 1.15, R2 = 0.97; BFPD-IA/ELISA 1.12, R2 = 0.95; BFPD-IA/ITA 0.95, R2 = 0.93. These very good performances and the agreement between BFPD-IA and ITA (routine diagnostic), combined with BFPD-IA's functional advantages over ITA (and ELISA)-such as quick time to result (~20 min), reduced consumed reagents (only one assay buffer and no washing), few and easy steps, and compatibility with nucleic-acid-amplification instruments-render it a potential approach for a reliable, cost-efficient, evidence-based point-of-care diagnostic test for guiding antibiotic prescriptions.

Au-X (X=Pt/Ru)-decorated magnetic nanocubes as bifunctional nanozyme labels in colorimetric, magnetically-enhanced, one-step sandwich CRP immunoassay

Scientific interest in the investigation and application of multifunctional nanomaterials in medical diagnostics has been increasing. The employment of magnetocatalytic immunoconjugates as both analyte-capturing agents and enzyme-like catalytic labels may enable rapid preconcentration and determination of relevant antigens. In this work, we synthesized and comprehensively characterized two types of noble metal-decorated magnetic nanocubes (MDMCs) which were subsequently applied in the one-step, sandwich nanozyme-linked immunosorbent assay (NLISA). Magnetic cores allow for rapid separation from complex samples of biological origin. The catalytically active shell composed of Au-decorated Pt or Ru can effectively mimic the activity of horseradish peroxididase, retaining at the same time the ability to form stable bioconstructs through self-assembly of thiolated ligands. As a result, hybrid multifunctional nanoparticles were synthesized and used to detect C-reactive protein (CRP) in serum samples. We have also paid considerable attention to the mechanistic studies of the formation of sandwich immunocomplexes with nanoparticle labels by means of immunoenzymatic methods and surface plasmon resonance. Analytical parameters of the Pt-MDMCs-labeled NLISA (detection limit LOD = 0.336 ng mL−1, recovery = 98.0%, linear response window covering two logarithmic units) turned out to be superior to the classical, one-step ELISA based on a horseradish peroxidase. In addition, our method offers further possibility of sensitivity adjustment by changing the parameters of magnetic preconcentration, together with good long-term stability of MDMCs conjugates and their good resistance to common interferences. We believe that the proposed simple synthetic protocol will guide a new approach to applying metal-decorated magnetic nanozymes as versatile and multifunctional labels for application in subsequent pre-analytical analyte concentration and immunoassays towards clinical applications.

A Randomized Controlled Trial of Dietary Supplementation with Prunes (Dried Plums) on Inflammatory Markers in Postmenopausal Women

The prevalence of osteoporosis among women aged 50 years and older is expected to reach 13.6 million by 2030. Osteoporosis is characterized by compromised bone strength due to reduced bone mineral density (BMD) and bone quality, representing a major public health issue that necessitates effective treatment regimens that are safe, cost‐effective, and associated with fewer adverse effects than pharmaceutical agents. Alternative dietary interventions such as prunes (dried plum) have been extensively studied to mitigate bone loss in preclinical models of osteoporosis. In postmenopausal women, estrogen deficiency triggers an upregulation of inflammatory pathways, which promotes bone loss, thus increasing risk of fractures. Our understanding of the effect of prune consumption on inflammatory markers in humans is limited, warranting further investigation. This study aimed to evaluate the effects of 12 months of prune consumption (two doses) on inflammatory mediators. Postmenopausal women (n=106, 55‐75 years old) with low BMD were randomized to consume 0g prunes/day (control), 50g prunes/day, or 100g prunes/day for 12 months. All participants received 1200mg calcium and 800 IU vitamin D3 as standard of care. We hypothesized that prune consumption at 50g/day and 100g/day will reduce inflammatory markers in postmenopausal women with low BMD compared to control group (0g/day). At baseline and after 12 months of prune consumption, serum and peripheral blood mononuclear cells (PBMCs) were isolated to quantify C‐reactive protein (CRP) using a high‐sensitivity CRP immunoassay, the number of circulating monocytes using flow cytometry, and pro‐inflammatory cytokines [interleukin (IL)‐1β, IL‐6, IL‐8, monocyte chemoattractant protein (MCP)‐1, and tumor necrosis factor (TNF)‐α] from lipopolysaccharide (LPS)‐stimulated PBMCs using a multiplex enzyme‐linked immunosorbent assay. Differences among groups were assessed using one‐way ANOVA or Kruskal‐Wallis test. No significant differences in baseline characteristics were found among the three treatment groups. Prune consumption did not alter serum CRP or the number of monocytes. However, consumption of 100g prunes/day resulted in a significant reduction from baseline in IL‐1β (p=0.013), IL‐6 (p=0.007), and IL‐8 (p=0.049) secretion and consumption of 50g prunes/day resulted in a significant reduction from baseline in TNF‐α (p<0.001) secretion from LPS‐stimulated PBMCs compared to the control group. Our findings demonstrate that 12 months of prune consumption suppresses inflammatory cytokines from LPS‐stimulated PBMCs. Thus, daily consumption of 50g‐100g of prunes may reduce inflammatory mediators that can contribute to bone loss in postmenopausal women.

Development of cellulosic material-based microchannel device capable of fluorescence immunoassay of microsamples.

Microfluidic immunoassay devices are a promising technology that can quickly detect biomarkers with high sensitivity. Recently, many studies implementing this technology on paper substrates have been proposed for improving cost and user-friendliness. However, these studies have identified problems with the large volume of sample required, low sensitivity, and a lack of quantitative accuracy and precision. In this paper, we report a novel structure implemented as a cellulosic material-based microchannel device capable of quantitative immunoassay using small sample volumes. We fabricated microfluidic channels between a transparent cellophane film and water-resistant paper to facilitate loading of small-volume samples and reagents, with a 40-μm-wide immunoreaction matrix constructed in the center of the microchannel using highly precise photolithography. A fluorescence sandwich immunoassay for C-reactive protein (CRP) was successfully implemented that required only a 1-μL sample volume and a 20-min reaction time. We confirmed that the limit of detection of the device was 10-20 ng/mL with a coefficient of variation under 5.6%, which is a performance level comparable to conventional plastic-based human CRP enzyme-linked immunosorbent assay (ELISA) kits. We expect that such devices will lead to the elimination of large amounts of medical waste from the use of ubiquitous diagnostics, a result that is consistent with environmental sustainability goals.

The effect of layer thickness and immobilization chemistry on the detection of CRP in LSPR assays

The immobilization of a capture molecule represents a crucial step for effective usage of gold nanoparticles in localized surface plasmon resonance (LSPR)-based bioanalytics. Depending on the immobilization method used, the resulting capture layer is of varying thickness. Thus, the target binding event takes place at different distances to the gold surface. Using the example of a C-reactive protein immunoassay, different immobilization methods were tested and investigated with regard to their resulting target signal strength. The dependency of the target signal on the distance to the gold surface was investigated utilizing polyelectrolyte bilayers of different thickness. It could be experimentally demonstrated how much the LSPR-shift triggered by a binding event on the gold nanoparticles decreases with increasing distance to the gold surface. Thus, the sensitivity of an LSPR assay is influenced by the choice of immobilization chemistry.

Bio-Functionalized Magnetic Nanoparticles for the Immunoassay of C-Reactive Protein and Procalcitonin in Cervicovaginal Secretions of Pregnant Women with Preterm Prelabor Rupture of Membranes to Predict Early-Onset Neonatal Sepsis.

PurposeEarly-onset sepsis is a major cause of neonatal morbidity and mortality. C-reactive protein (CRP) and procalcitonin (PCT) are acute phase reactants related to infection. The aim of this study was to explore the feasibility of measuring CRP and PCT concentrations in cervicovaginal secretions of pregnant women with preterm prelabor rupture of membranes (PPROM) using an immunomagnetic reduction (IMR) assay to predict early-onset neonatal sepsis.Patients and MethodsThis prospective study was performed at Mackay Memorial Hospital, Taipei, Taiwan from February 2015 to January 2018. Pregnant women with PPROM between 22 and 34 weeks of gestation were recruited. CRP and PCT concentrations in cervicovaginal secretions were measured using an IMR assay.ResultsThirty-five cervicovaginal secretion samples were obtained. After excluding two neonatal deaths, early-onset neonatal sepsis was diagnosed in 15 of the 33 surviving neonates. There was no significant relationship between cervicovaginal secretion CRP level and neonatal sepsis; however, cervicovaginal secretion PCT levels were significantly higher in the neonatal sepsis group than in the non-sepsis group (45.99 vs 9.54 ng/mL, P = 0.039). Receiver operating characteristic (ROC) curve analysis revealed a PCT cut-off level of 20.60 ng/mL to predict early-onset sepsis, and the area under the ROC curve was 0.71 (95% confidence interval 0.52 to 0.90, P = 0.039), with sensitivity and specificity of 73.3% and 77.8%, respectively.ConclusionMeasuring the concentration of PCT in cervicovaginal secretions with an IMR assay can predict early-onset sepsis in neonates born to mothers with PPROM.

Nets of biotin-derived gold nanoparticles as a label for the C-reactive protein immunoassay.

This study presents a promising approach for the one-pot generation of the biotin-derived gold nanoparticles (GNPs@biotin). We report a direct method for the reduction of tetrachloroauric acid with biotin and generation of the labels due to nets formed via biotin-streptavidin interactions. The synthesized GNPs@biotin have a characteristic plasmon maximum, excellent colloidal stability, and streptavidin coupling efficiency. The size of the GNPs@biotin:streptavidin nets and the efficiency of interaction with specific antibodies can be easily customized by the component concentrations and time of their interaction. Moreover, the proposed labels require no additional reagents or manipulations for the synthesis, separation, or purification. The developed labels were applied for the detection of the model antigen of C-reactive protein (CRP) as a major inflammation biomarker. The assembling labels demonstrated a competitive advantage limit of CRP detection (LOD) of 1.2ng/mL and a limit of quantification (LOQ) of 3.9ng/mL in human plasma comparable to classical immunoassays. Moreover, the proposed approach is universal and can be potentially applied for the quantitative determination of other biomarkers in a variety of immunoassays in a combination with specific biotinylated antibodies.

Peroxidase-mimicking nanozyme with surface-dispersed Pt atoms for the colorimetric lateral flow immunoassay of C-reactive protein.

Platinum-containing nanozymes with peroxidase-mimicking activity (PMA) have found a broad application in bioanalytical methods and are potentially able to compete with enzymes as the labels. However, traditionally used methods for the synthesis of nanozymes result in only a small fraction of surface-exposed Pt atoms, which participate in catalysis. To overcome this limitation, we propose a new approach for the synthesis of nanozymes with the efficient dispersion of Pt atoms on particles' surfaces. The synthesis of nanozymes includes three steps: the synthesis of gold nanoparticles (Au NPs), the overgrowth of a silver layer over Au NPs (Au@Ag NPs, 6 types of NPs with different thicknesses of Ag shell), and the galvanic replacement of silver with PtCl62- leading to the formation of trimetallic Au@Ag-Pt NPs with uniformly deposited catalytic sites and high Pt-utilization efficiency. Au@Ag-Pt NPs (23 types of NPs with different concentrations of Pt) with various sizes, morphology, optical properties, and PMA were synthesized and comparatively tested. Using energy-dispersive spectroscopy mapping, we confirm the formation of core@shell Au@Ag NPs and dispersion of surface-exposed Pt. The selected Au@Ag-Pt NPs were conjugated with monoclonal antibodies and used as the colorimetric and catalytic labels in lateral flow immunoassay of the inflammation biomarker: C-reactive protein (CRP). The colorimetric signal enhancement was achieved by the oxidation of 3,3'-diaminobenzidine by H2O2 catalyzed by Au@Ag-Pt NPs directly on the test strip. The use of Au@Ag-Pt NPs as the catalytic label produces a 65-fold lower limit of CRP detection in serum (15 pg mL-1) compared with Au NPs and ensures the lowest limit of detection for equipment-free lateral flow immunoassays. The assay shows a high correlation with data of enzyme-linked immunosorbent assay (R2 = 0.986) and high recovery (83.7-116.2%) in serum and plasma. The assay retains all the benefits of lateral flow immunoassay as a point-of-care method.

Application of Printer Toner As a Versatile Intermediate for Protein Immobilization in Flexible Immunosensing Platforms

Introduction For the last years, low-cost microfluidics and microarrays on polyester substrates have gained applications in a variety of fields, including analytical Po int-of- C are systems. The use of a 2D platforms brings a significant reduction in assay costs, both due to a lower consumption of materials and reagents, as well as a simplicity of manufacturing process in comparison to current approaches [1]. Commercial applicability of immunoassays requires the development of efficient strategies for antibody immobilization in bulk quantities on a wide variety of readily available substrates. Other factors, which influence the usefulness of antibody immobilization approach are scalability, reproducibility and cost-efficiency. Simple, one-step methods such as passive adsorption on hydrophobic surfaces or direct protein immobilization with the use of functionalized silanes are particularly appreciated in fabrication of immunosensing platforms [2]. The use of masking properties of cured toner as an intermediate for passive and covalent immobilization of antibodies on poly(ethylene terephtalate) (PET) seems to be an attractive and still undiscovered path for development of immunoassays on versatile, printable substrates [3]. Fabricatio n of t oner-based platforms for Ab-capturing an d immunosensing Throughout presented research two general approaches for immobilization of antibody and antibody-binding protein (protein A/G) were examined: 1) adsorption on pristine PET@toner foil via hydrophobic interactions, 2) amine-dependent, covalent binding on glycidyloxypropyl tromethoxysilane (GTPMS)- coated surface. Print pattern (at various grayscale levels) was designed in ChemSketch 2.0 and printed on PET surface using HP 100 LaserJet P1006 printer at resolution of 1200 dpi. Pristine PET@toner foils were used for passive adsorption. For GTPMS coating, hydrophilization of toner was performed by a 10-minute treatment with the use of UV/ozone cleaner. Then, 3% GTPMS solution in 50% ethanol was dispensed on freshly-oxidized foil for 30 min. Both multi-purpose substrates characterized by different surface properties were then incubated with rabbit anti-CRP antibody or protein A/G followed by surface blocking. Method To fully characterize the analytical performance in a role of immunosensing platforms, two types of immunoreactions were carried out on prepared 2D Ab/protein arrays. Direct immuno-labelling of rabbit anti-CRP antibody with anti-rabbit IgG-alkaline phosphatase (ALP) conjugate aimed at comparative evaluation of specificity and efficiency of antibody coating. Indirect sandwich immunoassay for C-reactive protein was selected as a model to determine basic working parameters of toner-based immunoassay. Antibody-capturing properties of protein A/G surface were evaluated by determination of monoclonal antibody-ALP conjugate binding capacity. For quantitative analysis, enzymatic reaction was carried out by dispensing p-nitrophenyl phosphate as chromogenic ALP substrate. Results and Conclusions Results of immunolabeling confirmed the usefulness of cured toner as an intermediate for efficient antibody and protein A/G coating via hydrophobicity-driven adsorption. Due to an increase of surface area and a hydrophobicity higher than for pristine PET, the densest layers were observed for fully printed surfaces (100% black level). The differences in the surface properties were also reflected in kinetics of antibody binding. UV/ozone treatment resulted in permanent oxidation and thus hydrophilization of toner surface. The introduction of hydroxyl groups favored reaction with GTPMS. Epoxy-silane coating of oxidized toner enabled a rapid fabrication of protein-reactive surface characterized by a high binding capacity in a single step immobilization. Thanks to good adhesion and thus tight bonding of laser-cut adhesive tape to PET@toner, the microchannel architecture was obtained. It opened up the possibility to carry out immunoassays in microfluidic systems. It was proven that presented methodologies, due to the design and fabrication simplicity, use of commonly available materials and up-scalability, could be successfully applied in immunodiagnostics or flexible microfluidics for Lab-on-a-Foil technology.

Inkjet Printing-Based Immobilization Method for a Single-Step and Homogeneous Competitive Immunoassay in Microchannel Arrays.

In this study, we report an inkjet printing-based method for the immobilization of different reactive analytical reagents on a single microchannel for a single-step and homogeneous solution-based competitive immunoassay. The immunoassay microdevice is composed of a poly(dimethylsiloxane) microchannel that is patterned using inkjet printing by two types of reactive reagents as dissolvable spots, namely, antibody-immobilized graphene oxide and a fluorescently labeled antigen. Since nanoliter-sized droplets of the reagents could be accurately and position-selectively spotted on the microchannel, different reactive reagents were simultaneously immobilized onto the same microchannel, which was difficult to achieve in previously reported capillary-based single-step bioassay devices. In the present study, the positions of the reagent spots and amount of reagent matrix were investigated to demonstrate the stable and reproducible immobilization and a uniform dissolution. Finally, a preliminary application to a single-step immunoassay of C-reactive protein was demonstrated as a proof of concept.

Coherent silicon photonic interferometric biosensor with an inexpensive laser source for sensitive label-free immunoassays.

Over the past two decades, integrated photonic sensors have been of major interest to the optical biosensor community due to their capability to detect low concentrations of molecules with label-free operation. Among these, interferometric sensors can be read-out with simple, fixed-wavelength laser sources and offer excellent detection limits but can suffer from sensitivity fading when not tuned to their quadrature point. Recently, coherently detected sensors were demonstrated as an attractive alternative to overcome this limitation. Here we show, for the first time, to the best of our knowledge, that this coherent scheme provides sub-nanogram per milliliter limits of detection in C-reactive protein immunoassays and that quasi-balanced optical arm lengths enable operation with inexpensive Fabry-Perot-type lasers sources at telecom wavelengths.

Metal-enhanced chemiluminescence detection of C-reaction protein based on silver nanoparticle hybrid probes

A novel metal-enhanced chemiluminescence (MEC) signal tag was designed. By combining with a disposable immunosensor chip, an ultrasensitive immunoassay method was proposed for detection of C-reactive protein (CRP), an essential cerebrovascular disease marker. Two kinds of silver nanoparticle (AgNP) probes, DNA-hemin/DNA-A/biotin-DNA modified AgNPs (Probe A) and DNA-hemin/DNA-B modified AgNPs (Probe B) were prepared respectively. The MEC signal tag was formed by the link of Probe A and Probe B through the hybridization of DNA-A and DNA-B. The formed AgNP hybrid probes brought excellent CL signal amplification, due to the increased content of hemin molecules and the great MEC effect. The AgNP hybrid probes can be bound to the biotinylated antibody of sandwich immunocomplex for immunoassay of CRP. Under optimal conditions, the method showed a wide detection range of 7 × 10−7 to 0.07 mg mL−1 and a detection limit down to 0.05 ng mL−1. The results obtained with clinical serum samples were in acceptable agreement with reference values. The AgNP hybrid probes as well as the MEC-based immunoassay method showed great potentials in early clinical diagnosis of cerebrovascular disease.

An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles

An origami paper-based electrochemical immunoassay for C-reactive protein (CRP) detection is described. The assay integrates multiple steps of electrode modification into a single device. A graphene-modified screen-printed carbon electrode (G/SPCE) was employed to enhance sensitivity. Gold nanoparticles were first electrodeposited onto the G/SPCE, followed by a self-assembled monolayer of L-cysteine. The capture anti-CRP was then covalently immobilized on the modified electrode. CRP was quantified by measuring the changes in the charge-transfer resistance of the electrode by using hexacyanoferrate as the redox probe. Cyclic voltammetry and scanning electron microscopy were also applied to verify the successful modification of the electrode. Under optimal conditions, impedance increase in the 0.05-100μgmL-1 CRP concentration range, and the limit of detection is 15ngmL-1 (at S/N = 3). The immunoassay was successfully applied to the determination of CRP in a certified human serum sample. This method is simple, low-cost, portable and disposable. Graphical abstract An origami paper-based analytical device (oPAD) is described that integrates the multistep of electrode modification, immobilization and detection into a single device. The direct conjugation between the capture antibody and target molecule was allowed to use in this system. The C-reactive protein (CRP) concentration in serum samples was determined using electrochemical impedance spectroscopy.

Silica-encapsulated quantum dots for highly efficient and stable fluorescence immunoassay of C-reactive protein

In this study, we presented a new coating regulation of silica by the reverse microemulsion method to obtain biocompatible high quality silica-encapsulated quantum dot (QD). The hydrophobic CdSe/ZnS QDs were coated with silica successfully and demonstrated good photostability and thermal stability and high fluorescent intensity. The corresponding silica-encapsulated QDs-antibody probes were used to detect C-reactive protein (CRP) with quantum dot-labeled immunosorbent assay (QLISA). Only micro dosage of probes was required due to good antibody-antigen combination in optimized buffer environment. The assay results are comparable to commercial assay kits with a wide analytical range of 1-1000 ng/mL, a low limit of detection of 0.76 ng/mL, and good recovery rates of 87.31–105.48%. The convenience and cost effective aspects of this silica-encapsulated QDs immunosorbent assay motivate future development of in vitro diagnostic kits.

Immunoassay of C-reactive protein by hot electron-induced electrochemiluminescence at polystyrene-carbon black composite electrodes

Aromatic Tb(III) chelates can be detected down to subnanomolar concentrations at polystyrene-carbon black composite electrodes on the basis of hot electron-induced electrochemiluminescence, and used as electrochemiluminescent labels in bioaffinity assays. The excitation mechanism is based on chemiluminescent reactions that are initiated by the field emission/tunnel emission of hot electrons from the composite electrode. The composition and the properties of the novel composite electrodes were studied in detail. Hot electron-induced electrochemiluminescence intensities obtainable with the present composite electrodes are comparable to the previously used metal/insulator-type electrodes without exhibiting considerable long-lived solid state electroluminescence background emission, unlike e.g. oxide-coated silicon and aluminum electrodes.C-reactive protein was finally determined in a heterogeneous sandwich-assay by using the present composite electrodes as a solid support material for the capture antibodies. The detection antibody was labeled with a commercially available Tb(II) chelate. Calibration curve for the determination of C-reactive protein was linear over two orders of magnitude and the detection limit was well below the clinical reference value, approx. 1 μg l−1. Disposable polystyrene-carbon black composite electrodes are superior alternatives for e.g. silicon-based electrodes due to the low cost, easier manufacturing process and a very good performance e.g. in immunoassays.

Label-Free Electrochemical Immunoassay for C-Reactive Protein.

C-reactive protein (CRP) is one of the most expressed proteins in blood during acute phase inflammation, and its minute level increase has also been recognized for the clinical diagnosis of cardio vascular diseases. Unfortunately, the available commercial immunoassays are labour intensive, require large sample volumes, and have practical limitations, such as low stability and high production costs. Hence, we have developed a simple, cost effective, and label-free electrochemical immunoassay for the measurement of CRP in a drop of serum sample using an immunosensor strip made up of a screen printed carbon electrode (SPE) modified with anti-CRP functionalized gold nanoparticles (AuNPs). The measurement relies on the decrease of the oxidation current of the redox indicator Fe3+/Fe2+, resulting from the immunoreaction between CRP and anti-CRP. Under optimal conditions, the present immunoassay measures CRP in a linear range from 0.4–200 nM (0.047–23.6 µg mL−1), with a detection limit of 0.15 nM (17 ng mL−1, S/N = 3) and sensitivity of 90.7 nA nM−1, in addition to a good reproducibility and storage stability. The analytical applicability of the presented immunoassay is verified by CRP measurements in human blood serum samples. This work provides the basis for a low-priced, safe, and easy-to-use point-of-care immunosensor assay to measure CRP at clinically relevant concentrations.

Membrane-based sample inlet for centrifugal microfluidic cartridges

Centrifugal microfluidics enables the rapid execution of complex blood sample analyses in a fully automated manner at the point of care. However, during blood sample addition, the cartridge is at rest and centrifugal forces are not present to allocate the sample in a controlled way. We present a versatile approach for the user-friendly, well-controlled and safe sample addition into a centrifugal microfluidic cartridge for use in serology. It features a commercial (plasma separation) membrane stacked into the inlet chamber of the cartridge. This combination of sample inlet and plasma separation in one structural unit brings the advantage of reducing footprint in highly integrated point-of-care testing. By using a pipette the user may add a blood sample (90μL) to the membrane holding the liquid by capillary forces. Flowing across the membrane, cells separate from blood plasma. The blood plasma releases into the downstream structure upon centrifugation. The mean plasma recovery rate was 57.3 (±4.7) % and mean plasma purity 99.5 (±0.6) % from samples of varying hematocrit (36%–59%). Furthermore, the analyte C-reactive protein (CRP) did not significantly adsorb to the membrane. This was concluded, since CRP immunoassay results with plasma from spiked whole blood, obtained from membrane-based plasma separation and from plasma separation on a standard laboratory centrifuge, did not significantly differ. Thus, the suggested approach is promising for simultaneous application as sample inlet holding the sample by capillary forces and as a plasma separation module for centrifugal microfluidics. Potential future applications may include other sample matrices. The use with further blood transfer devices (capillary, directly from fingertip) seems possible, yet requiring further evaluation.