Immunoassay Scheme Research Articles

Digital immunoassay for biomarker concentration quantification using solid-state nanopores

ABSTRACTSingle-molecule counting is the most accurate and precise method for determining the concentration of a biomarker in solution and is leading to the emergence of digital diagnostic platforms enabling precision medicine. In principle, solid-state nanopores—fully electronic sensors with single-molecule sensitivity—are well suited to the task. Here we present a digital immunoassay scheme capable of reliably quantifying the concentration of a target protein in complex biofluids that overcomes specificity, sensitivity, and consistency challenges associated with the use of solid-state nanopores for protein sensing. This is achieved by employing easily-identifiable DNA nanostructures as proxies for the presence (“1”) or absence (“0”) of the target protein captured via a magnetic bead-based sandwich immunoassay. As a proof-of-concept, we demonstrate quantification of the concentration of thyroid-stimulating hormone from human serum samples down to the high femtomolar range. Further optimization to the method will push sensitivity and dynamic range, allowing for development of precision diagnostic tools compatible with point-of-care format.

Quantum Plasmonic Immunoassay Sensing.

Plasmon-polaritons are among the most promising candidates for next-generation optical sensors due to their ability to support extremely confined electromagnetic fields and empower strong coupling of light and matter. Here we propose quantum plasmonic immunoassay sensing as an innovative scheme, which embeds immunoassay sensing with recently demonstrated room-temperature strong coupling in nanoplasmonic cavities. In our protocol, the antibody-antigen-antibody complex is chemically linked with a quantum emitter label. Placing the quantum-emitter-enhanced antibody-antigen-antibody complexes inside or close to a nanoplasmonic (hemisphere dimer) cavity facilitates strong coupling between the plasmon-polaritons and the emitter label resulting in signature Rabi splitting. Through rigorous statistical analysis of multiple analytes randomly distributed on the substrate in extensive realistic computational experiments, we demonstrate a drastic enhancement of the sensitivity up to nearly 1500% compared to conventional shifting-type plasmonic sensors. Most importantly and in stark contrast to classical sensing, we achieve in the strong-coupling (quantum) sensing regime an enhanced sensitivity that is no longer dependent on the concentration of antibody-antigen-antibody complexes down to the single-analyte limit. The quantum plasmonic immunoassay scheme thus not only leads to the development of plasmonic biosensing for single molecules but also opens up new pathways toward room-temperature quantum sensing enabled by biomolecular inspired protocols linked with quantum nanoplasmonics.

Disposable Electrochemical Immunosensor Based on Dual-Labeled Gold Nanoprobe (Ab-Au-HRP) for Detection of Alpha-Fetoprotein

A disposable electrochemical immunosensor for detection of human alpha-fetoprotein (AFP) has been developed in this study, using monoclonal antibody against AFP and horseradish peroxidase co-modified colloidal gold nanoparticles (Ab-Au-HRP) as trace label and screen-printed carbon electrodes (SPCEs) as signal transducers. The immunosensor was based on a sandwich immunoassay scheme in which the nanoprobe Ab-Au-HRP was dispensed on the polyester membrane as conjugate pad. The other monoclonal antibody against AFP, as capture antibody, was immobilized onto the nitrocellulose membrane which was mounted on the carbon surface of the screen-printed electrodes. An absorbent paper was mounted downstream to draw the sample to successively flow through the conjugate, capture and absorbent pads. The resulting sandwich immune reaction was evaluated by electrochemical detection, through addition of a single-component substrate for HRP. Under optimum conditions, the current value was linearly increased with an increased AFP concentration from 0.5 to 350 ng/mL, at a relatively low detection limit of 0.25 ng/mL at 3 sigma. Excellent analytical performance, fabrication reproducibility and operational stability for the proposed immunosensor indicated its promising application in clinical diagnostics and self-exams.

Observation of Brownian relaxation of magnetic nanoparticles using HTS SQUID

We analyzed the sensitivity of a separationless immunoassay scheme using functionalized magnetic nanoparticles (MNPs) and a sensitive HTS SQUID magnetometer. The signal of a 100 mu L sample at a concentration of 1 mg/mL and field of 7.5 nT was 20 m Phi(0). This makes it possible for the sensitivity to be within the range of 50 ng/mL at the required time of up to 100 s per a point in the frequency spectrum.

Sensitive Cytokine Assay Based on Optical Fiber Allowing Localized and Spatially Resolved Detection of Interleukin-6.

We demonstrated a cytokine detection device based on gold nanoparticle modified silica optical fiber for the monitoring of locally variable cytokine interleukin-6 (IL-6) concentrations using a sandwich immunoassay scheme. The fiber is designed to be introduced into an intrathecal catheter with micrometer-sized holes drilled along its length to enable fluid exchange between the outside and inside of the catheter. An exposed optical fiber (diameter 125 μm) modified with a layer of gold nanoparticles was functionalized with the IL-6 capture antibody to form the sensing interface. The immunocapture device was incubated with a cytokine solution to capture the analyte. The device was then exposed to the IL-6 detection antibody which was loaded on the fluorescently labeled magnetic nanoparticles, making it possible to quantify the cytokine concentration based on the intensity of fluorescence. A reliable method for quantifying the fluorescent signal on a 3D structure was developed. This device was applied to the detection of cytokine IL-6 with the low limit of detection of 1 pg mL-1 in a sample volume of 1 μL. The device has the linear detection range of 1-400 pg mL-1 and spatial resolution on the order of 200-450 μm, and it is capable of detecting localized IL-6 secreted by live BV2 cells following their liposaccharide stimulation. This biological detection system is suitable for monitoring multiple health conditions.

A photoacoustic immunoassay for biomarker detection

Challenges in protein biomarker analysis include insufficient sensitivity for detecting low-abundance biomarkers, poor measurement reproducibility, and the high costs and large footprints of detection systems. To address these issues, a new detection modality was developed for analyzing protein biomarkers based on the plasmon-enhanced photoacoustic (PA) effect. The detection modality employed a heterogeneous immunoassay scheme and used gold nanoparticles (AuNPs) as the signal reporter. Due to their localized plasmon resonance, AuNPs can strongly interact with intensity-modulated laser excitation and generate strong PA signals, which are subsequently sensed and quantified using a microphone. As an example, the performance of the PA immunoassay was evaluated by detecting the human interleukin 8 chemokine. The PA immunoassay provided approximately 143× lower limit of detection (LOD) than observed with the gold standard enzyme-linked immunosorbent assay – a decrease from 23pg/mL to 0.16pg/mL. In addition to the significant performance improvement in terms of the LOD, the PA immunoassay also offers advantages in terms of compatibility with low-cost instruments and the long-term stability of assay results.

A competitive microfluidic immunological clenbuterol analysis using a microELISA system

We present a novel method to analyze clenbuterol based on a competitive microfluidic immunoassay scheme with a micro-ELISA system, and obtain a limit of detection that is less than 0.1 ng ml−1 with a quantitative working range of 0.1 ng ml−1 to 27.0 ng ml−1. The approach was envisaged to be a promising method for efficient onsite clenbuterol control with good sensitivity and portability.

An Ultrasensitive Simultaneous Multianalyte Immunoassay Based on Arsenic and Mercury Ions Labeled SiO2@Au Nanoparticle Probes

Abstract An ultrasensitive immunoassay was developed on the basis of As3+ and Hg2+ labeled SiO2@Au nanoparticles (NPs) probe and hydride generation-atomic fluorescence spectrometry (HG-AFS) for the detection of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Firstly, amino SiO2@Au NPs were synthesized for selective absorption of As3+ and Hg2 to form As(Hg)-SiO2@Au NPs signal tags, respectively. Subsequently, the prepared probes were labeled on the secondary antibody (Ab2) of CEA and CA 19-9, respectively. Based on the sandwich immunoassay scheme, corresponding Ab1, antigen and Ab2 modified with probes, were conjugated to form the immunocomplex on the bottom of 96-well plate at room temperature. After the immunocomplex were dissolved in an alkali solution, As3+ and Hg2+ were released into the solution and detected by HG-AFS, the atomic fluorescence intensity of As3+ and Hg2+ were proportional with logarithms of CEA and CA 19-9 concentration, then indirect quantitative analysis of CEA and CA 19-9 was realized. This assay was based on the determination of the concentration of As3+ and Hg2+ by HG-AFS for quantization of the corresponding CEA and CA 19-9 antigen. The assay showed a wide linear range of 0.001–100.0 µg L−1 for CEA and 0.01–80 U mL−1 for CA 19-9, with the detection limit of 0.5 ng L−1 and 0.005 U mL−1, respectively. This method was used in the determination of real serum samples, and the results were consistent with that from ELISA. This assay showed three orders of magnitude of sensitivity higher than that of ELISA, which provided promising simultaneous multianalyte immunoassays (SMIAs) in the early diagnosis of pancreatic cancer.

Au-labeled antibodies to enhance the sensitivity of a refractometric immunoassay: Detection of cocaine

An integrated platform for a very sensitive detection of cocaine based on a refractometric biosensor is demonstrated. The system uses a waveguide grating biosensor functionalized with a cocaine multivalent antigen-carrier protein conjugate. The immunoassay scheme consists of the competitive binding of cocaine-specific antibodies to the immobilized conjugates. A 1000-fold enhancement of the sensor's sensitivity is achieved when using gold conjugated monoclonal antibodies instead of free antibodies. Together with the optimization of the assay conditions, the setup is designed for a quick identification of narcotics using automated sampling. The results show that the presence of cocaine in a liquid sample could be identified down to a concentration of 0.7nM within one minute. This value can be reduced even further when longer binding time is allowed (0.2nM after 15min). Application of the system to detection of narcotics at airport security control points is discussed.

Electrical Readout of Protein Microarrays on Regular Glass Slides

A new approach for the electrical readout of microarrays prepared on regular glass slides, using an array of impedimetric transducers (interdigitated electrodes, IDEs) is presented in this work. Impedance detection relies on the use of a urease-labeled immunoassay scheme. Urease is able to produce an increase in conductivity by hydrolysis of the urea substrate, which is measured with the IDEs and directly related to the amount of target analyte. Unlike previous electrical microarrays, the assay does not take place on top of the transducers but on a regular glass slide, which may enable the development of compact multiplexed analytical systems with lower cost per assay. A droplet of solution with the enzymatic substrate is deposited on each transducer of the array, and the microarray is positioned at a short distance (300 μm) so that each droplet wets one transducer and one spot of the microarray. This procedure allows reusing the transducer array for readout of a virtually unlimited number of microarrays. A microarray based on an immunoassay for the detection of a mouse generic protein in a concentration range from 0.03 to 30 μg mL(-1) was carried out to assess the performance of the electrical readout approach. A sigmoid response with a limit of detection of 0.1 μg mL(-1) and a dynamic range of 1 order of magnitude was obtained. A comparative study was also carried out with two well established analytical procedures. First, the urease-based immunoassay was tested in a 96 well microtiter plate using phenol red pH indicator and absorbance detection. Second, the microarray was carried out using the same target protein concentration range but applying a Cy3 label and fluorescence detection. Both assays allowed for the validation of the performance of the presented electrical readout system.

Single-wall carbon nanotube forest arrays for immunoelectrochemical measurement of four protein biomarkers for prostate cancer.

Protein arrays that measure multiple protein cancer biomarkers in clinical samples hold great promise for reliable early cancer detection. Herein, we report a prototype 4-unit electrochemical immunoarray based on single-wall carbon nanotube forests for the simultaneous detection of multiple protein biomarkers for prostate cancer. Immunoarray procedures were designed to measure prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interleukin-6 (IL-6) simultaneously in a single serum sample. All of these proteins are elevated in serum of patients with prostate cancer, but they have widely different relative levels of serum concentration. Horseradish peroxidase (HRP) was used as label on detection (secondary) antibodies in a sandwich immunoassay scheme. Biotinylated secondary antibodies (Ab(2)) that bind specifically to streptavidin-HRP conjugates provided 14-16 labels per antibody and gave the necessary higher sensitivity required for PF-4 and IL-6 detection at physiological levels. Conventional singly labeled Ab(2)-HRP conjugates were sufficient for PSA and PSMA detection. Immunoarrays were used to measure four biomarkers in clinical human serum samples of prostate cancer patients and controls with excellent correlation to referee enzyme-linked immunosorbent (ELISA) assays.

Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 μg L −1 and EC 50 0.079 μg L −1 were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase. In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection. Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.

Development of the microchip-based repeatable immunoassay system for clinical diagnosis

A microchip-based semi-automated heterogeneous immunoassay was developed wherein a flow injection-like system and repeatable-use gel were employed. Microfluidics used a pump with highly accurate control of flow rate, two injector valves and a microchip. The results of a system suitability test based on dye injection for microfluidics including a thermal lens microscope as a detector were good. A regeneration buffer for antigen–antibody interaction of brain natriuretic peptide (BNP) was found which did not lose binding activity of the antibody on the gels and integration of such an immunoassay scheme into a semi-automated microfluidics system would facilitate repeated-use heterogeneous immunoassay. Good reproducibility of BNP assay was obtained with the selected regeneration buffer. The concentration of BNP in the range of 0.1–100 pg mL−1 (n = 3) was successfully determined using the system and protocol developed here.

Immunochemical determination of sulfamethazine in river water and medicinal preparations

A new variant of the immunochemical assay for determining sulfamethazine, an antimicrobial drug belonging to the class of sulfonamide derivatives, has been developed based on the fluorescence polarization technique. The immunoassay is carried out using a polyclonal antiserum against several sulfonamides (conjugated with bovine serum albumin) and a fluorescent label (tracer). The proposed fluorescence polarization immunoassay ensures sulfamethazine detection with a threshold of 6 ng/ml and has a dynamic range of linearity for drug concentrations from 0.05 to 25.7 µg/ml. The proposed method has been applied to the determination of sulfamethazine in river water and tablets. Various methods of tablet pretreatment using methanol, acetonitrile, potassium hydroxide solution, and hydrochloric acid have been tried and compared. The accuracy of the immunoassay was confirmed by the recovery test. The possibility of using the obtained antiserum and the proposed immunoassay scheme for the determination of some other sulfonamides is demonstrated.

A disposable immunomagnetic electrochemical sensor based on functionalised magnetic beads and carbon-based screen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls (PCBs)

A disposable immunomagnetic electrochemical sensor involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed and applied for the detection of polychlorinated biphenyls (PCBs). The sensor was based on a direct competitive immunoassay scheme in which antibody-coated magnetic beads were used as solid phase; then SPCEs were employed as transducers for the evaluation of the extent of the immunochemical reaction using an alkaline phosphatase (AP) labelled tracer. The α-naphthyl phosphate was used as enzymatic substrate and the α-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). A detection limit of 0.4 ng/mL for Aroclor 1248 PCB mixture was obtained. The performance of the sensor was successfully evaluated on marine sediment extracts and soil samples spiked with different Aroclor mixtures indicating that this new and sensitive technique offers great promise for decentralized environmental applications.

Rapid Amperometric Immunoassay for Escherichia coli Based on Graphite Coated Nylon Membranes

AbstractAn amperometric immunosensor for rapid detection of bacteria has been developed. A graphite‐coated nylon membrane serves as a support for antibody immobilization and as a working electrode. The different parameters affecting sensor preparation such as, selection of the conductive material, pore size of the nylon membrane, number of coatings and the ratios of the graphite to the binder were optimized. A “sandwich” immunoassay scheme was used and amperometric detection of captured bacteria was conducted by the electro‐reduction of iodine formed as the product of a peroxidase catalyzed reaction. This approach has been used for detection of total Escherichia coli with a low detection limit of 40 cells/mL. The developed system can be easily adapted for rapid analysis of different pathogenic microorganisms.

ICP-MS-based competitive immunoassay for the determination of total thyroxin in human serum

To further expand the range of analytes that can be detected by using ICP-MS coupled with bioanalytical methods, we have employed a new separation system based on the highly active surface streptavidin and biotinylated monoclonal antibody (McAb) in a competitive immunoassay followed by ICP-MS detection. Specifically, we have demonstrated its application for the determination of total thyroxine (T4) in human serum using Eu3+ as a label. In this method, streptavidin immobilized to the pre-coated bovine serum albumin (BSA)-biotin on the microwells showed a significantly higher binding capacity for the biotinylated anti-T4 monoclonal antibody. Total T4 was quantified by measuring the Eu3+ intensity with ICP-MS after using 1% HNO3 to extract Eu3+ from the bound fraction of the immunocomplex. The detection limit was 7.4 ng mL−1 with a sample volume of 25 µL. Total T4 values obtained by this procedure agreed well with those obtained by a chemiluminescent immunoassay method. Both inter- and intra-assay precisions were below 10%. The results indicate that a general competitive ICP-MS-based immunoassay scheme may be possible.

Fiber optic-based biosensor for ricin

We report on an evanescent wave fiber-optic biosensor for detecting a potently toxic protein, ricin, in the picograms per milliliter range. A sandwich immunoassay scheme was used to detect ricin. First, an anti-ricin IgG was immobilized onto the surface of an optical fiber in two different ways. In the first method, the antibody was directly coated to the silanized fiber using a crosslinker. Second, avidin-coated fibers were incubated with biotinylated anti-ricin IgG to immobilize the antibody using an avidin–biotin bridge. The assay using the avidin–biotin linked antibody demonstrated higher sensitivity and wider linear dynamic range than the assay using antibody directly conjugated to the surface. The linear dynamic range of detection for ricin in buffer using the avidin–biotin chemistry is 100 pg/ml–250 ng/ml. The limits of detection for ricin in buffer solution and river water are 100 pg/ml and 1 ng/ml, respectively. At higher concentrations of ricin (>50 ng/ml), we observe a strong interaction of ricin with the avidin coated on the surface of the fibers. We have demonstrated that this interaction is primarily due to the lectin activity of ricin and is significantly reduced using fibers coated with neutravidin or by adding galactose to the ricin samples.