A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching.

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

Development of a Fluorescent Microfluidic Device Based on Antibody Microarray Read-Out for Therapeutic Drug Monitoring of Acenocoumarol.

The development of a proof-of-concept point-of-care (PoC) device for the determination of oral anticoagulants determination is presented. Acenocoumarol (ACL) is prescribed to prevent certain cardiovascular diseases related to the prevention of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Oral anticoagualant treatment (OAT) represents a population of 2% under treatment which has expenditures about $ 144 million in 2011. The main drawback for OAT is the associated narrow therapeutic window and the unpredictable dose-response relationship, which is one of the main causes for visiting the emergency room at the hospitals. In a previous work, family antibodies were produced for the simultaneous detection of ACL and warfarin (W) depending on the area of application. It was developed in different formats, indirect and direct, either with similar detectabilities and both assays quantifying the oral anticoagulants with high accuracy and reproducibility. We present the implementation of the already developed immunochemical method to a point-of-care (PoC) device to assist on the patient compliance assessment programs. In order to achieve this goal, a first development was performed implementing ACL ELISA assay into a microarray format with fluorescent read-out. The assay was successfully implemented achieving a LOD of 1.23 nM of ACL directly measured in human plasma. Then, a fully integrated microfluidic system is developed which incorporates the specific immunoreagents for the detection of ACL. The immunoreagents were attached onto the glass slide in a microarray format. The system is automatic, rapid, sensitive, and disposable that could help clinicians monitor patients under OAT. According to the fluorescent label of the ACL binding, the chip can be easily read with a scanner. The microfluidic system performed good according to the robust and reproducible signals, and subsequently yielded an accurate result.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching / 药物分析学报

Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal en-ables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concen-tration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3％and 110.8％with relative standard deviations of 7.3％-17.6％,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.

Enhanced Bio-Barcode Immunoassay Using Droplet Digital PCR for Multiplex Detection of Organophosphate Pesticides.

A bio-barcode immunoassay based on droplet digital polymerase chain reaction (ddPCR) was developed to simultaneously quantify triazophos, parathion, and chlorpyrifos in apple, cucumber, cabbage, and pear. Three gold nanoparticle (AuNP) probes and magnetic nanoparticle (MNP) probes were prepared, binding through their antibodies with the three pesticides in the same tube. Three groups of primers, probes, templates, and three antibodies were designed to ensure the specificity of the method. Under the optimal conditions, the detection limits (expressed as IC10) of triazophos, parathion, and chlorpyrifos were 0.22, 0.45, and 4.49 ng mL-1, respectively. The linear ranges were 0.01-20, 0.1-100, and 0.1-500 ng mL-1, and the correlation coefficients (R2) were 0.9661, 0.9834, and 0.9612, respectively. The recoveries and relative standard deviations (RSDs) were in the ranges of 75.5-98.9 and 8.3-16.7%. This study provides the first insights into the ddPCR for the determination of organophosphate pesticides. It also laid the foundation for high-throughput detection of other small molecules.

ASSURED Point-of-Need Food Safety Screening: A Critical Assessment of Portable Food Analyzers.

Standard methods for chemical food safety testing in official laboratories rely largely on liquid or gas chromatography coupled with mass spectrometry. Although these methods are considered the gold standard for quantitative confirmatory analysis, they require sampling, transferring the samples to a central laboratory to be tested by highly trained personnel, and the use of expensive equipment. Therefore, there is an increasing demand for portable and handheld devices to provide rapid, efficient, and on-site screening of food contaminants. Recent technological advancements in the field include smartphone-based, microfluidic chip-based, and paper-based devices integrated with electrochemical and optical biosensing platforms. Furthermore, the potential application of portable mass spectrometers in food testing might bring the confirmatory analysis from the laboratory to the field in the future. Although such systems open new promising possibilities for portable food testing, few of these devices are commercially available. To understand why barriers remain, portable food analyzers reported in the literature over the last ten years were reviewed. To this end, the analytical performance of these devices and the extent they match the World Health Organization benchmark for diagnostic tests, i.e., the Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users (ASSURED) criteria, was evaluated critically. A five-star scoring system was used to assess their potential to be implemented as food safety testing systems. The main findings highlight the need for concentrated efforts towards combining the best features of different technologies, to bridge technological gaps and meet commercialization requirements.

A plasmonic biosensor array exploiting plasmon coupling between gold nanorods and spheres for domoic acid detection via two methods.

An immunoassay was developed that utilized plasmonic coupling between immobilised gold nanorods and colloid gold nanospheres to detect the marine toxin domoic acid (DA). The aspect ratio of the nanorods was optimised and the effects of variation in acidity, silver to gold ratio, cetyltrimethylammonium bromide (CTAB) concentration and seed addition in the growth solution on the yield, size variance and LSPR peak position was investigated. Excellent nanorods (size variation < 15%; aspect ratio 3.5-5; yield 0.26-0.35 nM mL-1) were obtained for the LSPR range 785-867 nm using strong acidic conditions (12 µl HCl (37%)), silver to gold ratio of 1:5, 0.05-0.1 M CTAB and 20-30 µl seed addition to 10 mL of growth solution. One set of nanorods (54.9 X 15.7 nm; LSPR 785 nm) were immobilised onto a silica support and bio-functionalised with DA hapten. Colloid nanospheres (15 nm; LSPR 519 nm) were bio-functionalised with an anti-domoic-acid monoclonal antibody. The functionalised nanoparticles were used to detect DA by plasmon coupling by quantifying the average LSPR shift of individual plasmon couples with hyperspectral imaging or quantifying the pixels count caused by the particle aggregation visible under darkfield microscopy. The first method led to a LSPR blue-shift of ~55 nm caused by the immunoreaction. The second, simpler method, enabled very clear qualitative detection (p < 0.0005) of domoic acid when 10 µM domoic acid was added. Both methods show potential though the novelty and simplicity of the second platform allowing rapid (~30 min) detection with high-throughput possibilities using a simple set-up is of most interest.

Multiplexed Immunosensor Based on the Amperometric Transduction for Monitoring of Marine Pollutants in Sea Water.

Environmental pollutants vigilance is one of the main problems that the aquaculture industry has to face with the objective to ensure the quality of their products and prevent entrance in the food chain that finally may arrive to the consumer. Contaminants such as hormones, antibiotics or biocides are especially relevant due to their toxicity, pharmacological effect or hormonal activity that can be considered harmful for the final consumer. The contaminants can be detected in the environment where the food is growing, and their concentration can be found (i.e., seawater) in the range of µg·L-1, ng·L-1 or even in lower concentrations. Thus, sensitive and selective methods for their monitoring are required to avoid their arrival in the food chain. Here, the development of a multiplexed amperometric biosensor is described, based on the use of specific antibodies to reach the necessary detectability to measure the targeted contaminants directly in seawater. The multiplexed immunosensor allows the detection of four relevant pollutants, such as el Irgarol 1051, sulfapyridine, chloramphenicol and estradiol, reaching an IC50 of 5.04 ± 0.29, 3.45 ± 0.29, 4.17 ± 0.44 and 5.94 ± 0.28 µg·L-1, directly measured in seawater.

Competitive ELISA for N-terminal pro-brain natriuretic peptide (NT-proBNP) determination in human plasma.

Brain natriuretic peptides (BNPs) are well-established cardiovascular disease (CVD) biomarkers that are released from the heart after ventricular wall stress. Particularly, the N-terminal proBNP (NT-proBNP) is a 76 aa long peptide and is recognized as an indicator for the diagnosis of heart failure (HF) and is being used in routine tests in emergency rooms when levels are above 0.4 ng mL-1. Herein, we report a new competitive ELISA for NT-proBNP, which is able to detect this biomarker directly in undiluted human plasma samples. The ELISA has been the result of a rational design of an immunizing peptide hapten and the investigation of different immunochemical conditions, including heterologous competitors and distinct physico-chemical conditions. The developed ELISA is able to detect NT-proBNP with a LOD of 0.40 ± 0.15 ng mL-1 in human plasma samples and quantify this biomarker in the range between 0.97 ± 0.38 and 23.10 ± 9.46 ng mL-1 with good accuracy. The ELISA can simultaneously measure many samples in 1.5 h and has been found to be robust, reproducible and shows great promise in diagnosis of heart failures.

Development of Novel Magneto-Biosensor for Sulfapyridine Detection.

In this work, we report the development of a highly sensitive biosensor for sulfapyridine detection based on an integrated bio micro-electromechanical system (Bio-MEMS) containing four gold working electrodes (WEs), a platinum counter electrode (CE), and a reference electrode (RE). Firstly, the cleaned WEs were modified with 4-aminophenylacetic acid (CMA). Then, (5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2BSA) was immobilized onto the transducers surface by carbodiimide chemistry. The analyte was quantified by competitive detection with SA2BSA immobilized on the WE toward a mixture of Ab155 antibody (with fixed concentration) and sulfapyridine. In order to obtain a highly sensitive biosensor, Ab155 was immobilized onto magnetic latex nanoparticles surface to create a 3D architecture (Ab-MLNp). Using electrochemical impedance spectroscopy (EIS), we investigated the influence of the Ab-MLNp on the sensitivity of our approach. The optimized system was analyzed, as competitive assay, with different concentrations of sulfapyridine (40 µM, 4 µM, and 2 nM) and with phosphate buffer solution. From data fitting calculations and graphs, it was observed that the EIS showed more linearity when Ab-MLNp was used. This result indicates that the magnetic latex nanoparticles increased the sensitivity of the biosensor.

The benefits of carbon black, gold and magnetic nanomaterials for point-of-harvest electrochemical quantification of domoic acid.

Gold nanostars (GNST), gold nanospheres (GNP) and carbon black (CB) are chosen as alternative nanomaterials to modify carbon screen-printed electrodes (c-SPEs). The resulting three kinds of modified c-SPEs (GNP-SPE, CB-SPE and GNSP-SPE) were electrochemically and microscopically characterized and compared with standardized c-SPEs after pretreatment with phosphate buffer by pre-anodization (pre-SPE). The results show outstanding electrochemical performance of the carbon black-modified SPEs which show low transient current, low capacitance and good porosity. A competitive chronoamperometric immunoassay for the shellfish toxin domoic acid (DA) is described. The performances of the CB-SPE, GNP-SPE and pre-SPE were compared. Hapten-functionalized magnetic beads were used to avoid individual c-SPE functionalization with antibody while enhancing the signal by creating optimum surface proximity for electron transfer reactions. This comparison shows that the CB-SPE biosensor operated best at a potential near - 50 mV (vs. Ag/AgCl) and enables DA to be determined with a detection limit that is tenfold lower compared to pre-SPE (4 vs. 0.4 ng mL-1). These results show very good agreement with HPLC data when analysing contaminated scallops, and the LOD is 0.7 mg DA kg-1 of shellfish. Graphical abstractSchematic representation of the magnetic bead-based immunoassay for the quantification of domoic acid (DA) in shellfish with nanomaterial-modified screen-printed electrodes. CB, carbon black; GNP, gold nanospheres; GNST, gold nanostars; MB, magnetic beads; DA-mAb, anti-DA monoclonal mouse antibody; HRP-pAb, horseradish conjugated polyclonal goat anti-mouse antibody; DA-BSA, bovine serum albumin conjugated DA; HQ, hydroquinone; BQ, benzoquinone.

Development and validation of a multianalyte immunoassay for the quantification of environmental pollutants in seawater samples from the Catalonia coastal area.

Five different enzyme-linked immunosorbent assays (ELISAs) have been developed and applied for the detection of five representatives of important families of chemical pollutants in seawater: Irgarol 1051® (triazine biocide), sulfapyridine and chloramphenicol (antibiotics), 17ß-estradiol (hormone), and domoic acid (algae toxin). The assays were validated by high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (HRMS) showing good correlation between both immunochemical and chemical techniques. A process of extraction and clean-up was added prior to the analysis based on solid-phase extraction (SPE). The multianalyte platform presented good specificity for each compound and adequate sensitivity, with limits of detection (LOD) after the SPE treatment of 0.124 ± 0.006, 0.969 ± 0.09, 0.20 ± 0.05, 1.11 ± 0.012, and 1.39 ± 0.09 ng L-1 for Irgarol 1051®, sulfapyridine, chloramphenicol, 17ß-estradiol, and domoic acid, respectively. No matrix effects were noticed in working with the seawater extracts. Afterward, seawater samples from the Mediterranean Sea (coastal area of Catalonia) were analyzed by both techniques and only one sample presented one contaminant, 17ß-estradiol, in the concentration of 0.011 ± 0.04 µg L-1.

Nanobody: outstanding features for diagnostic and therapeutic applications.

Nanobodies (Nbs) have arisen as an alternative to conventional antibodies (Abs) and show great potential when used as tools in different biotechnology fields such as diagnostics and therapy. Different approaches have been described for the production of Nbs and these methods face new challenges focused on improving yield, affinity, and reducing production costs. This review summarizes these challenges, and also the latest advances in the detection of different kinds of molecules, such as proteins and small molecules, and describes their potential use for noninvasive in vivo imaging and for in vitro assays. Moreover, the unique properties of Nbs are outlined like internalization, size, thermal and chemical stability, affinity, blood clearance, and labeling procedures. Concerning therapeutic applications, we highlight some already reported examples about Nbs being used for the treatment of several diseases such as cancer, neurodegenerative or infectious diseases among others. Finally, future trends, opportunities, and disadvantages are also discussed.

Light-induced mechanisms for nanocarrier's cargo release.

Nanomaterials have been the focus of attention in several fields, including biomedicine, electronics, or catalysis, mainly due to the novel properties of the materials at the nanoscale. In the field of diagnosis, nanomaterials have been contemplated as an opportunity to improve sensitivity and time of response, therefore, facilitating early treatment and monitoring of the disease. For therapeutic applications, new drug delivery nanosystems aiming to provide enhanced efficiency have been proposed often addressing selective or controlled delivery of therapeutic agents to particular cells to maximize treatment efficacy minimizing adverse effects. The therapeutic agents can be dissolved, adsorbed, entrapped, encapsulated or attached on the surface or inside the nanocarriers. Given the context of the different generations of nanocarriers and their wide range of applications, the present article aims to discuss the nature of external stimuli which will trigger the controlled release of different biomolecules. For each class, a brief description of the physical principle, basic concepts, as well as some examples, are reported. A final discussion focused on the real implications and needs for optimal drug delivery system is presented, altogether with some considerations and prospects in the trends that diagnostics applications could follow in the next years.

Studies towards hcTnI Immunodetection Using Electrochemical Approaches Based on Magnetic Microbeads.

Different electrochemical strategies based on the use of magnetic beads are described in this work for the detection of human cardiac troponin I (hcTnI). hcTnI is also known as the gold standard for acute myocardial infarction (AMI) diagnosis according to the different guidelines from the European Society of Cardiology (ESC) and the American College of Cardiology (ACC). Amperometric and voltamperometric sandwich magnetoimmunoassays were developed by biofunctionalization of paramagnetic beads with specific antibodies. These bioconjugates were combined with biotinylated antibodies as detection antibodies, with the aim of testing different electrochemical transduction principles. Streptavidin labeled with horseradish peroxidase was used for the amperometric magnetoimmunoassay, reaching a detectability of 0.005 ± 0.002 µg mL-1 in 30 min. Cadmium quantum dots-streptavidin bioconjugates were used in the case of the voltamperometric immunosensor reaching a detectability of 0.023 ± 0.014 µg mL-1.

Nanoplasmonic biosensor device for the monitoring of acenocoumarol therapeutic drug in plasma.

Acenocoumarol (Sintrom®) is an oral anticoagulant prescribed for the treatment of a variety of thromboembolic disorders such as atrial fibrillation and thrombosis or embolism. It inhibits fibrin production preventing clot formation. Acenocoumarol has a narrow therapeutic range, and its effects depend on several factors, such as body weight, age, metabolism, diet, certain medical conditions or the intake of additional drugs, among others. A higher dose may result in the risk of bleeding, while if it is too low, the risk of blood clot can increase. Complementary tools that allow the therapeutic drug monitoring (TDM) of acenocoumarol plasmatic levels from the starting of the treatment would be of paramount importance to personalize the treatment. Point-of-care (POC) devices can offer an added value in facilitating on-site monitoring (i.e. hospitals, primary care doctor or even by the patient itself) and can aid in dosage management. With this aim, we have developed a compact and simple nanoplasmonic sensing device based on gold nanodisks for the rapid monitoring of acenocoumarol, using highly specific polyclonal antibodies produced against this drug. A specific and reproducible label free indirect competitive assay has been developed and the viability of performing the evaluation directly in plasma diluted 1:1 has been demonstrated. A limit of detection (LOD) of only 0.77 ±â€¯0.69 nM, an IC50 of 48.2 ±â€¯5.12 nM and a dynamic range between 3.38 ±â€¯1.33 nM and 1154 ±â€¯437 nM were achieved, which easily fit within the drug plasma levels of acenocoumarol, making this approach a highly attractive option for its decentralized monitoring in human plasma.

Immunoassay and amperometric biosensor approaches for the detection of deltamethrin in seawater.

The study of an enzyme-linked immunosorbent assay (ELISA) and an amperometric biosensor for the detection of the pyrethroid deltamethrin in seawater is reported. The preparation of specific polyclonal antibodies is addressed using two immunizing haptens based on deltamethrin and cypermethrin compounds, with a spacer arm placed at the cyano residue in the pyrethroid structure. Different conjugates based on bovine serum albumin and aminodextran are prepared depending on the lipophilic profile of the competitor haptens studied. A reproducible and sensitive indirect competitive ELISA is developed, reaching a limit of detection of 1.2 ± 0.04 µg L-1 and an IC50 value of 21.4 ± 0.3 µg L-1 (both n = 3). For validation of the assays described, artificial seawater samples fortified with deltamethrin are analyzed. For the ELISA assay, these accuracy studies reported a slope of 0.904. An amperometric immunosensor is developed using the same immunoreagents and achieving a comparable detectability in terms of LOD of 4.7 µg L-1, measuring seawater without any pretreatment. These results suggest that both techniques can be used as rapid and simple analytical methods for deltamethrin quantification in seawater samples, which are great candidates for initial environmental screening programs. Graphical abstract ᅟ.

Enzyme-linked immunosorbent assays for therapeutic drug monitoring coumarin oral anticoagulants in plasma.

The development of high-throughput immunochemical assays to assist on precision medicine for patients treated with coumarin oral anticoagulants (OA) is reported. The assays are able to quantitate Warfarin (W) and/or Acenocoumarol (ACL) directly in plasma samples without any previous sample pretreatment. The detectabilities (W, 3.52 ±â€¯2.25 nM and ACL, 1.56 ±â€¯0.64 nM) and the working ranges achieved (W, 1.19 ±â€¯0.73 to 12.05 ±â€¯2.99 nM and ACL 0.63 ±â€¯0.20 to 10.19 ±â€¯6.69 nM) are within the therapeutic levels usually found in patients treated with these drugs. The assays are specific with only cross-recognition of 4'-NH2-ACL on the ACL ELISA, which is one of the main metabolites of this drug. Moreover, accuracy studies performed with blind spiked samples show very good correlation between the spiked and the measured concentrations. Finally, a small clinical pilot study has been performed analyzing 96 plasma samples from treated and untreated patients, showing that the assay is able to quantitate ACL. The results obtained allow envisaging the possibility to use these assays for pharmacokinetic studies, dosage assessment or therapeutic drug monitoring.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples.

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17ß-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19 ±â€¯0,06 µg L-1; sulfapyridine, 0.17 ±â€¯0.07 µg L-1; chloramphenicol, 0.11 ±â€¯0.03 µg L-1; BDE-47, 2.71 ±â€¯1.13 µg L-1; 17ß-estradiol, 0.94 ±â€¯0.30 µg L-1 and domoic acid, 1.71 ±â€¯0.30 µg L-1). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was < 20% in all the cases. No sample pre-treatment was necessary, and the results could be obtained in just 1 h 30 min. The microarray shows high sample throughput capabilities, being able to measure simultaneously more than 68 samples and screen them for a significant number of chemical contaminants of interest in environmental screening programs.

New approach based on immunochemical techniques for monitoring of selective estrogen receptor modulators (SERMs) in human urine.

Antiestrogenic compounds such as tamoxifen, toremifen and chlomifen are used illegally by athletes to minimize physical impacts such as gynecomastia resulting from the secondary effects of anabolic androgenic steroids, used to increase athletic efficiency unlawfully. The use of these compounds is banned by the World Anti-Doping Agency (WADA) and controls are made through analytical methodologies such as HPLC-MS/MS, which do not fulfil the sample throughput requirements. Moreover, compounds such as tamoxifen are also used to treat hormone receptor-positive breast cancer (ER + ).Therapeutic drug monitoring (TDM) of tamoxifen may also be clinically useful for guiding treatment decisions. An accurate determination of these drugs requires a solid phase extraction of patient serum followed by HPLC-MS/MS. In the context of an unmet need of high-throughput screening (HTS) and quantitative methods for antiestrogenic substances we have approached the development of antibodies and an immunochemical assay for the determination of these antiestrogenic compounds. The strategy applied has taken into consideration that these drugs are metabolized and excreted in urine as the corresponding 4-hydroxylated compounds. A microplate-based ELISA procedure has been developed for the analysis of these metabolites in urine with a LOD of 0.15, 0.16 and 0.63 µg/L for 4OH-tamoxifen, 4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL established by WADA (20 µg/L).

Biobarcode assay for the oral anticoagulant acenocoumarol.

A novel approach for therapeutic drug monitoring of oral anticoagulants (OA) in clinical samples is reported, based on a NP-based biobarcode assay. The proposed strategy uses specific antibodies for acenocumarol (ACL) covalently bound to magnetic particles (pAb236-MP) and a bioconjugate competitor (hACL-BSA) linked to encoded polystyrene probes (hACL-BSA-ePSP) on a classical competitive immunochemical format. By using this scheme ACL can be detected in low nM range (LOD, 0.96 ± 0.26, N = 3, in buffer) even in complex samples such as serum or plasma (LOD 4 ± 1). The assay shows a high reproducibility (%CV 1.1 day-to-day) and is robust, as it is demonstrated by the fact that ACL can be quantified in complex biological samples with a very good accuracy (slope = 0.97 and R2 = 0.91, of the linear regression obtained when analyzing spiked vs measured values). Moreover, we have demonstrated that the biobarcode approach has the potential to overcome one of the main challenges of the multiplexed diagnostic, which is the possibility to measure in a single run biomarker targets present at different concentration ranges. Thus, it has been proven that the signal and the detectability can be modulated by just modifying the oligonucleotide load of the encoded probes. This fact opens the door for combining in the same assay encoded probes with the necessary oligonucleotide load to achieve the detectability required for each biomarker target.