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.

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.

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.

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.

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.

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.

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.

Lipoprotein(a) determination in human serum using a nitrilotriacetic acid derivative immunosensing scaffold on disposable electrodes.

A novel strategy for the construction of a disposable integrated amperometric immunosensor for the sensitive and rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum is reported. The approach uses a sandwich format involving the covalent immobilization of selective capture antibodies (antiLp(a)) on the surface of N-[Nα,Nα-bis(carboxymethyl)-lysine]-12-mercaptododecanamide (HS-NTA)-modified screen-printed carbon electrodes (SPCEs). After a blocking step with skimmed milk, the modified antiLp(a)-SPCEs were incubated with a mixture solution containing the target analyte and a fixed concentration of a specific biotinylated antibody (biotin-antiLp(a)) and a streptavidin-horseradish peroxidase (HRP) (Strep-HRP) conjugate. The amperometric responses of the resulting immunosensor at -0.10 V (vs an Ag pseudo-reference electrode), upon addition of 3,3',5,5'-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate, were used to monitor the extent of the immunoreactions. The developed methodology exhibited a wide range of linearity between 0.02 and 10 µg mL(-1), a low detection limit (LOD) of 8 ng mL(-1), and a great selectivity against other serum components. The usefulness of the Lp(a) immunosensor was demonstrated by analyzing spiked serum samples as well as a reference serum containing a certified Lp(a) content.

Development and ELISA Characterization of Antibodies against the Colistin, Vancomycin, Daptomycin, and Meropenem: A Therapeutic Drug Monitoring Approach.

More than 70% of bacteria are resistant to all or nearly all known antimicrobials, creating the need for the development of new types of antimicrobials or the use of "last-line" antimicrobial therapies for the treatment of multi-resistant bacteria. These antibiotics include Glycopeptide (Vancomycin), Polymyxin (Colistin), Lipopeptide (Daptomycin), and Carbapenem (Meropenem). However, due to the toxicity of these types of molecules, it is necessary to develop new rapid methodologies to be used in Therapeutic Drug Monitoring (TDM). TDM could improve patient outcomes and reduce healthcare costs by enabling a favorable clinical outcome. In this way, personalized antibiotic therapy emerges as a viable option, offering optimal dosing for each patient according to pharmacokinetic (PK) and pharmacodynamic (PD) parameters. Various techniques are used for this monitoring, including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and immunoassays. The objective of this study is the development and characterization by ELISA of specific polyclonal antibodies for the recognition of the antibiotics Vancomycin (glycopeptide), Colistin (polymyxin), Daptomycin (lipopeptide), and Meropenem (carbapenem) for future applications in the monitoring of these antibiotics in different fluids, such as human plasma. The developed antibodies are capable of recognizing the antibiotic molecules with good detectability, showing an IC50 of 0.05 nM for Vancomycin, 7.56 nM for Colistin, 183.6 nM for Meropenem, and 13.82 nM for Daptomycin. These antibodies offer a promising tool for the precise and effective therapeutic monitoring of these critical antibiotics, potentially enhancing treatment efficacy and patient safety.

Synthesis of steroid-oligonucleotide conjugates for a DNA site-encoded SPR immunosensor.

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N(1)up, N(2)up, and N(3)up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid-oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid-oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid-oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).

Multiplexed immunoassay to detect anabolic androgenic steroids in human serum.

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community.

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.

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.

Preliminary study for simultaneous detection and quantification of androgenic anabolic steroids using ELISA and pattern recognition techniques.

A first step towards the multidetection, identification and quantification of anabolic androgenic steroids by enzyme-linked immunosorbent assay (ELISA) has been performed in this study. This proposal combines multiple competitive ELISA assays with different cross-reactivity profiles and multivariate data analysis techniques. Data have been analyzed by principal component analysis in conjunction with a novel K-nearest line classifier. This proposal allows simultaneous detection of up to four different steroids in the range of concentration from 0.1 to 316.2 nM with a total rate of 90.6% of correct detection, even in the presence of cross-reactivities. A methodology for concentration prediction is also presented with satisfactory results.

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.

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.

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.

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.

Nonlinear immunofluorescent assay for androgenic hormones based on resonant structures.

We report for the first time the use of two photon fluorescence as detection method of affinity binding reactions. We use a resonant grating waveguide structure as platform enhancement for detecting the interaction between fluorescent labeled Boldenone, a non-natural androgenic hormone, and a specific anti-anabolic antibody. We were able to detect a surface coverage of approximately 0.7 ng/mm(2).