An Optimized Bioassay for Mucin1 Detection in Serum Samples

Two simple and sensitive electrochemical approaches for Mucin1 (MUC1) tumor marker using magnetic beads coupling screen‐printed arrays were developed. The single‐use bioassays are based on a sandwich format in which aptamers or antibodies were coupled respectively to Streptavidin or Protein G‐modified magnetic beads. The bioreceptor‐modified beads are used to capture the MUC1 protein from the sample and sandwich assay is performed by the addition of a labeled secondary aptamer or antibody. The enzyme alkaline phosphatase and its substrate (1‐naphthyl phosphate) are then used for the electrochemical detection by differential pulse voltammetry (DPV). The analytical performances of the designed bioassays were compared in terms of sensitivity, selectivity and reproducibility. Using the optimized conditions, a linear range from 0 to 0.28 nM was obtained, with 0.19 nM LOD using antibody‐based and 0.07 nM LOD using aptamer‐based sandwich assay in MUC1 buffered solutions. The results also showed that the aptamer‐based approach exhibited higher selectivity for MUC1, allowing the detection of the protein in complex matrices. The developed aptasensor for MUC1 detection was applied on serum samples obtained from cancer patients, providing promising perspectives for clinical applications.     

Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

A fast, simple and sensitive bioelectrochemical magnetic immunosensing method is developed to monitor a potential insecticide biomarker, trichloropyridinol (TCP), in environmental sample. A magnet/glassy carbon (MGC) working electrode was used to accumulate immunocomplex associated magnetic beads and separate free and unbound reagents after liquid phase competitive immunoreaction among TCP antibody coated magnetic beads, TCP analyte and horseradish peroxidase (HRP) labeled TCP. The activity of HRP tracers was monitored by square‐wave voltammetry (SWV) by scanning electrocactive enzymatic product in the presence of 3,3′,5,5′‐tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB‐H₂O₂) substrate solution. The electrochemical signal of enzymatic product was greatly enhanced by dual accumulation events: magnetic accumulation of enzyme tracers bound magnetic beads and constant potential accumulation of enzymatic product. The voltammetric characteristics of substrate and enzymatic product were investigated, and the parameters of SWV analysis and immunoassay were optimized. Under the optimal conditions the immunosensor was used to measure as low as 5 ng L^?1 (ppt) TCP, which is 50‐fold lower than the value indicated by the manufacture of the TCP RaPID Assay kit (0.25 μg/L, colorimetric detection). The performance of the developed immunosensing system was successfully evaluated with river water samples spiked with TCP, indicating this convenient and sensitive technique offers great promise for decentralized environmental application. This technique could be readily used for detection of other environmental contaminants by developing specific antibodies against the contaminants and are expected to open new opportunities for environmental monitoring and public health.     

Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system

This study reports a new microfluidic system with three integrated functional devices for pumping, mixing and separation of bio-samples by utilizing micro-electro-mechanical-systems technology. By using antibody-conjugated magnetic beads, the developed system can be used to purify and enrich virus samples such that the subsequent detection of viruses can be performed with a higher sensitivity. The target viruses were first captured by the antibody coated onto the magnetic beads by using a rotary micromixer which performed the incubation process. The viruses were then purified and enriched by a magnetic field generated by planar microcoils. The integrated microfluidic system can perform the whole purification and enrichment process automatically using a rotary micropump and appropriate microvalves. In addition, a numerical simulation was also employed to optimize the design of the microcoils and to investigate the magnetic field strength and distribution. The simulation results were consistent with experimental observations. Finally, the developed system was used to successfully perform the purification and enrichment of Dengue viruses. The detectable limit of Dengue viruses was found to be as low as 10(2) pfu ml(-1) by using this approach. Therefore, the integrated microsystem can perform incubation, transportation, mixing and purification of virus samples, possibly making it a promising platform for future biological and medical applications.     

Preparation and structural evolution of SiO(2)-TiO(2) pillared layered manganese oxide nanocomposite upon intercalating reaction

Poly(ethylene glycol) possessing pentaethylenehexamine at one end (N6-PEG) was prepared via a reductive amination reaction of aldehyde-ended PEG with pentaethylenehexamine. Using N6-PEG, an antibody/PEG co-immobilized surface was constructed on magnetic particles via an active ester reaction method. After immobilization of the antibody on the active ester surface, N6-PEG was reacted on the magnetic beads. A sandwich enzyme-linked immunosorbent assay (ELISA) system was newly constructed using PEG/antibody co-immobilized magnetic beads combined with an alkaline phosphatase (ALP)-assisted fluorescent detection system using alpha-fetoprotein (AFP) as a model antigen. The co-immobilization of both antibody and PEG on the magnetic bead surfaces reduced the nonspecific adsorption of proteins from cell lysates. Especially, when the magnetic particle surface was modified by N6-PEG mixtures with different molecular weights of 6000 and 2500 (6 kDa:2.5 kDa=9:1 w/w), the nonspecific adsorption of proteins was strongly suppressed. It is rather surprising for us that the sensitivity of the antibody on the surface was enhanced significantly when the PEG tethered chain was constructed in between the surface antibodies. Consequently, the mixed N6-PEG treatment showed a much higher S/N ratio than for the corresponding beads treated with bovine serum albumin (BSA), a conventional blocking reagent. Actually, when alpha-fetoprotein was analyzed by the magnetic bead-assisted ELISA thus constructed, the S/N ratio was about 20-fold higher for the mixed coating with PEG (6 kDa):PEG (2.5 kDa)=9:1, compared to the conventional BSA.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.     

Development of a novel label-free amperometric immunosensor for the detection of okadaic acid

Okadaic acid (OA), a lipophilic phycotoxin is mainly produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, a novel experimental methodology for label-free detection of OA was developed. Protein G magnetic beads (protein-G-MBs) modified gold electrode was used to immobilize anti-OA monoclonal antibody (anti-OA-MAb). Preliminary, colorimetric tests were performed in order to validate protein-G-MBs and anti-OA-MAb reaction. Electrochemical detection was carried out by differential pulse voltammetry in ferri/ferrocyanide solution. The limit of detection value obtained (0.5 μg L⁻¹) validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed with real samples, showing a good percentage of recovery.     

Biometallization‐Based Electrochemical Magnetoimmunosensing Strategy for Avian Influenza A (H7N9) Virus Particle Detection

A highly sensitive electrochemical immunosensor for avian influenza A (H7N9) virus (H7N9 AIV) detection was proposed by using electrochemical magnetoimmunoassay coupled with biometallization and anodic stripping voltammetry. This strategy could accumulate the enzyme‐generated product on the surface of the magneto electrode by means of silver deposition, which amplified the detection signal about 80 times. The use of magnetic beads (MBs) and the magneto electrode could also amplify the detection signal. Furthermore, a bi‐electrode signal transduction system was introduced into this immunosensor, which is also beneficial to the immunoassay. A concentration as low as 0.011 ng mL^?1 of H7N9 AIV could be detected in about 1.5 h with good specificity. This study not only provides a simple and sensitive approach for virus detection but also offers an effective signal enhancement strategy for the development of highly sensitive MB‐based electrochemical immunoassays.     

Quantum-dots based electrochemical immunoassay of interleukin-1α

We describe a quantum-dot (QD, CdSe@ZnS) based electrochemical immunoassay to detect a protein biomarker, interleukin-1α (IL-1α). QD conjugated with anti-IL-1α antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary IL-1α antibody (immobilized on the avidin-modified magnetic beads), IL-1α, and the QD-labeled secondary antibody, QD labels were attached to the magnetic-bead surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured QDs was used to quantify the concentration of IL-1α after an acid-dissolution step. The streptavidin-modified magnetic beads and the magnetic separation platform were used to integrate a facile antibody immobilization (through a biotin/streptavidin interaction) with immunoreactions and the isolation of immunocomplexes from reaction solutions in the assay. The voltammetric response is highly linear over the range of 0.5–50 ng ml⁻¹ IL-1α, and the limit of detection is estimated to be 0.3 ng ml⁻¹ (18 pM). This QD-based electrochemical immunoassay shows great promise for rapid, simple, and cost-effective analysis of protein biomarkers.     

Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.     

Development of an Electrochemical Immunoassay Based on the Use of an Eight‐Electrodes Screen‐Printed Array Coupled with Magnetic Beads for the Detection of Antimicrobial Sulfonamides in Honey

In this work an electrochemical immunoassay, based on a direct competitive assay, was developed using magnetic beads as solid phase and carbon screen‐printed arrays as transducers for the detection of sulfonamides in food matrices such as honey. Magnetic beads coated with protein A were modified by immobilisation of specific antibodies and then the competition between the target analyte and the corresponding analyte‐labelled with an enzyme was carried out; after the immunosensing step, beads were captured by a magnet onto the working surfaces of a screen‐printed eight‐electrodes array for a multiple electrochemical detection. Screen‐printed eight‐electrodes arrays were chosen as transducers due to the possibility to repeat multiple analysis and to test different samples simultaneously. Alkaline Phosphatase (AP) was used as enzyme label and Differential Pulse Voltammetry (DPV) as fast electrochemical technique. Calibration curves demonstrate that the developed electrochemical immunoassay was able to detect this class of drugs in standard solutions at low concentrations (ng/mL levels). The short incubation times (25 min) and the fast electrochemical measurement (10 sec) make of these systems a possible alternative to classic ELISA tests.     

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 EC₅₀ 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.     

Aptamer based strategy for cytosensing and evaluation of HER-3 on the surface of MCF-7 cells by using the signal amplification of nucleic acid-functionalized nanocrystals

The electrochemical detection of cell lines of MCF-7 (human breast cancer) has been reported, using magnetic beads for the separation tool and high-affinity DNA aptamers for signal recognition. The high specificity was obtained by using the magnetic beads and aptamers, and the good sensitivity was realized with the signal amplification of DNA capped CdS or PbS nanocrystals. The ASV (anodic stripping voltammetry) technology was employed for the detection of cadmic cation and lead ions, for electrochemical assay of the amount of the target cells and biomarkers on the membrane of target cells, respectively. This electrochemical method could respond to as low as 100 cells mL⁻¹ of cancer cells with a linear calibration range from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹, showing very high sensitivity. Moreover, the amounts of HER-3 which were overexpressed on MCF-7 cells were calculated correspond to be 3.56 × 10⁴ anti-HER-3 antibody molecules. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamers and magnetic beads used in the assay, indicating the wide applicability of the assay for early and accurate diagnose of cancers.     

Rapid automatic nucleic acid purification system based on gas–liquid immiscible phase

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas–liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%–95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.     

Electrochemical Magnetoimmunosensing Approach for the Sensitive Detection of H9N2 Avian Influenza Virus Particles

A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2 avian influenza virus particles (H9N2 AIV) was designed based on the combination of high‐efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin–streptavidin system. The reusable, homemade magneto Au electrode (M‐AuE) was designed and used for the direct sensing. Immunocomplex‐coated magnetic beads (IMBs) were easily accumulated on the surface of the M‐AuE to obtain the catalytically reduced electrochemical signal of H₂O₂ after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic‐bead‐based electrochemical immunosensor showed better analytical performance than the planar‐electrode‐based immunosensor with the same sandwich construction. Amounts as low as 10 pg mL^?1 H9N2 AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.     

Polychlorinated biphenyls (PCBs) detection in milk samples by an electrochemical magneto-immunosensor (EMI) coupled to solid-phase extraction (SPE) and disposable low-density arrays

An electrochemical immunosensor for polychlorinated biphenyl (PCB) detection based on graphite screen-printed low-density arrays and on magnetic beads is reported.The immunological reaction for the detection of PCBs is based on a direct competitive assay using alkaline phosphatase (AP) as enzymatic label. After the immunochemical recognition, the modified magnetic beads are captured by a magnet on the surface of the graphite working electrode. The electrochemical detection is thus achieved through the addition of the AP substrate (α-naphthyl-phosphate).Two different antibodies (sIgG anti-PCB28 and rIgG anti-PCB77) were tested and compared in terms of sensitivity and ability to recognise different congeners. The developed electrochemical magneto-immunosensor (EMI) was successfully combined with solid-phase extraction (SPE) for the analysis of PCBs in milk samples. In spiked samples a recovery of 80% was obtained. The proposed strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of clinical, food and environmental samples, considering also that low-density arrays allow the simultaneous analysis of different processed samples.     

Sensitive immunoassay for porcine pseudorabies antibody based on fluorescence signal amplification induced by cation exchange in CdSe nanocrystals

We report on a novel immunoassay for porcine pseudorabies virus (PRV) antibody that is based on fluorescence signal amplification induced by silver(I) ion exchange in CdSe nanocrystals. An antigen-antibody-secondary antibody sandwich structure was first formed from PRV, PRV antibody, and CdSe-labeled rabbit anti-pig antibody. Then, the Cd(II) ions in the CdSe labels were released by a cation exchange reaction with Ag(I). Released Cd(II) was finally quantified using the sensitive fluorescent probe Rhodamine 5 N. Due to this signal amplification, the sensitivity and linear range of the immunoassay were largely improved (compared to the traditional ELISA) in having a limit of detection as low as 1.2 ng?mL^?1 of PRV antibody and a linear range from 2.44 to 312 ng?mL^?1. The successful determination of PRV antibody in pig serum samples is proof for the utility of the method.

Electrochemical Sensing of Ovalbumin Based on the Interaction between Lysozyme Origin/Tyrosine‐rich Peptides Modified on Magnetic Beads and Oligothreonine/Ovalbumin‐origin Peptide

We developed a highly sensitive electrochemical system for the sensing of ovalbumin (OVA). Lysozyme origin/tyrosine‐rich peptides (RNRCKGTDVQAWY₄C) were immobilized on magnetic beads, and the competitive reaction between OVA and oligothreonine/OVA origin peptide probe (T₈VLLPDEVSG) could then be measured. In a previous study, the detection of OVA at the 10^?13 M level was achieved using RNRCKGTDVQAWY₄C‐modified beads via a cross‐linker. To improve the sensitivity to OVA, this system uses T₈VLLPDEVSG peptide probe to measure the interaction to RNRCKGTDVQAWY₄C immobilized on magnetic beads. The peak of Y₄C actually was an electron‐transfer peptide, which represented the oxidation of a phenolic hydroxyl group. First, we confirmed that the oxidation response of Y₄C was increased based on an improvement in the electron transfer accessibility by oligothreonine. Next, T₈VLLPDEVSG peptide probe was used for the electrochemical sensing of OVA in solutions that contained consistent amounts of RNRCKGTDVQAWY₄C on magnetic beads. As a result, the peak current decreased as the concentration of OVA increased. The sensitivity to OVA was improved compared with the use of only RNRCKGTDVQAWY₄C on magnetic beads. The OVA detection level was 10^?14 M, which approximates the results from antibody‐antigen reactions. Consequently, the proposed system is a powerful new concept in protein sensing.     

Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads

A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm of the concentration of APnEOs was plotted against the chemiluminescence intensity as the number of photons in 100 ms using standard APnEOs sample solutions at various concentrations (0–1000 ppb) under optimum conditions. The lower detection limit defined as IC₈₀ is ca 10 ppb. The time required for analysis is less than 15 min per a sample. The present method was successfully applied to the determination of APnEOs in river water.     

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL⁻¹) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL⁻¹.     

Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum

Two different D‐dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid‐modified magnetic beads (COOH‐MBs) and screen‐printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at ?0.20 V upon addition of H₂O₂, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL^?1 D‐dimer). The sandwich configuration has been successfully tested with serum samples.