Micromotor-based electrochemical immunoassays for reliable determination of amyloid-ß (1-42) in Alzheimer's diagnosed clinical samples.

Alzheimer's disease (AD), in addition to being the most common cause of dementia, is very difficult to diagnose, with the 42-amino acid form of Aß (Aß-42) being one of the main biomarkers used for this purpose. Despite the enormous efforts made in recent years, the technologies available to determine Aß-42 in human samples require sophisticated instrumentation, present high complexity, are sample and time-consuming, and are costly, highlighting the urgent need not only to develop new tools to overcome these limitations but to provide an early detection and treatment window for AD, which is a top-challenge. In recent years, micromotor (MM) technology has proven to add a new dimension to clinical biosensing, enabling ultrasensitive detections in short times and microscale environments. To this end, here an electrochemical immunoassay based on polypyrrole (PPy)/nickel (Ni)/platinum nanoparticles (PtNPs) MM is proposed in a pioneering manner for the determination of Aß-42 in left prefrontal cortex brain tissue, cerebrospinal fluid, and plasma samples from patients with AD. MM combines the high binding capacity of their immunorecognition external layer with self-propulsion through the catalytic generation of oxygen bubbles in the internal layer due to decomposition of hydrogen peroxide as fuel, allowing rapid bio-detection (15 min) of Aß-42 with excellent selectivity and sensitivity (LOD = 0.06 ng/mL). The application of this disruptive technology to the analysis of just 25 µL of the three types of clinical samples provides values concordant with the clinical values reported, thus confirming the potential of the MM approach to assist in the reliable, simple, fast, and affordable diagnosis of AD by determining Aß-42.

Micromotor-based dual aptassay for early cost-effective diagnosis of neonatal sepsis.

Given the long-life expectancy of the newborn, research aimed at improving sepsis diagnosis and management in this population has been recognized as cost-effective, which at early stages continues to be a tremendous challenge. Despite there is not an ideal-specific biomarker, the simultaneous detection of biomarkers with different behavior during an infection such as procalcitonin (PCT) as high specificity biomarker with one of the earliest biomarkers in sepsis as interleukin-6 (IL-6) increases diagnostic performance. This is not only due to their high positive predictive value but also, since it can also help the clinician to rule out infection and thus avoid the use of antibiotics, due to their high negative predictive value. To this end, we explore a cutting-edge micromotor (MM)-based OFF-ON dual aptassay for simultaneous determination of both biomarkers in 15 min using just 2 µL of sample from low-birth-weight neonates with gestational age less than 32 weeks and birthweight below 1000 g with clinical suspicion of late-onset sepsis. The approach reached the high sensitivities demanded in the clinical scenario (LODPCT = 0.003 ng/mL, LODIL6 = 0.15 pg/mL) with excellent correlation performance (r > 0.9990, p < 0.05) of the MM-based approach with the Hospital method for both biomarkers during the analysis of diagnosed samples and reliability (Er < 6% for PCT, and Er < 4% for IL-6). The proposed approach also encompasses distinctive technical attributes in a clinical scenario since its minimal sample volume requirements and expeditious results compatible with few easy-to-obtain drops of heel stick blood samples from newborns admitted to the neonatal intensive care unit. This would enable the monitoring of both sepsis biomarkers within the initial hours after the manifestation of symptoms in high-risk neonates as a valuable tool in facilitating prompt and well-informed decisions about the initiation of antibiotic therapy.These results revealed the asset behind micromotor technology for multiplexing analysis in diagnosing neonatal sepsis, opening new avenues in low sample volume-based diagnostics.

On the Move-Sensitive Fluorescent Aptassay on Board Catalytic Micromotors for the Determination of Interleukin-6 in Ultra-Low Serum Volumes for Neonatal Sepsis Diagnostics.

A graphene oxide/nickel/platinum nanoparticle micromotor (MM)-based fluorescent aptassay is proposed to determine interleukin-6 (IL-6) in serum samples from low-birth-weight infants (gestational age of less than 32 weeks and birthweight below 1000 g) with sepsis suspicion. In this kind of patients, IL-6 has demonstrated good sensitivity and specificity for the diagnosis of sepsis, both for early and late onset sepsis. The approach was based on the adsorption of the aptamer for IL-6 tagged with 6-FAM as a fluorescent label (AptIL-6, λem = 520 nm) on the graphene oxide external layer (MMGO-AptIL-6) inducing fluorescence quenching (OFF state) and a subsequent on-the-move affinity recognition of IL-6 from AptIL-6 (IL-6-AptIL-6 complex) recovering the fluorescence (ON state). An aptamer against IL-6 was selected and developed by the systematic evolution of ligands by exponential enrichment technology. This approach displayed a suitable linear range of 0.07-1000 pg mL-1 (r = 0.995) covering the cut-off and clinical practice levels, allowing direct determination without any dilution and simplifying the analysis as well as exhibiting an excellent sensitivity (LOD = 0.02 pg mL-1) in ultralow volumes of diagnostic clinical samples (2 µL). A high agreement between IL-6 levels obtained from our MM-based approach and the method used by the Hospital was obtained (relative error < 3%). The MM-based aptassay is competitive in comparison with that of the Hospital, in terms of a significant reduction of the sample volume (15 times less) and enhanced sensitivity, employing similar analysis times. These results position MM technology with enough potential to achieve high sensitivities in low sample volumes, opening new avenues in diagnosis based on low sample volumes.

Magnetic Bead-Based Electrochemical Immunoassays On-Drop and On-Chip for Procalcitonin Determination: Disposable Tools for Clinical Sepsis Diagnosis.

Procalcitonin (PCT) is a known protein biomarker clinically used for the early stages of sepsis diagnosis and therapy guidance. For its reliable determination, sandwich format magnetic bead-based immunoassays with two different electrochemical detection approaches are described: (i) disposable screen-printed carbon electrodes (SPE-C, on-drop detection); (ii) electro-kinetically driven microfluidic chips with integrated Au electrodes (EMC-Au, on-chip detection). Both approaches exhibited enough sensitivity (limit of detection (LOD) of 0.1 and 0.04 ng mL-1 for SPE-C and EMC-Au, respectively; cutoff 0.5 ng mL-1), an adequate working range for the clinically relevant concentrations (0.5-1000 and 0.1-20 ng mL-1 for SPE-C and EMC-Au, respectively), and good precision (RSD < 9%), using low sample volumes (25 µL) with total assay times less than 20 min. The suitability of both approaches was successfully demonstrated by the analysis of human serum and plasma samples, for which good recoveries were obtained (89-120%). Furthermore, the EMC-Au approach enabled the easy automation of the process, constituting a reliable alternative diagnostic tool for on-site/bed-site clinical analysis.

Polymer-Based Micromotor Fluorescence Immunoassay for On-the-Move Sensitive Procalcitonin Determination in Very Low Birth Weight Infants' Plasma.

A new fluorescence micromotor-based immunoassay (FMIm) has been developed for procalcitonin (PCT) determination as an early sepsis diagnostic analytical tool. The micromotors combine the high binding capacity of the specific antibodies onto their polymeric polypyrrole outer layer (PPy layer), with their magnetic guidance (Ni layer) and self-propulsion by catalytic generation of oxygen bubbles (PtNP inner layer) to actively recognize the PCT antigen. This FMIm allowed a sensitive (LOD = 0.07 ng mL-1) and direct PCT determination in clinical samples from very low-birth-weight infants (VLBWI) with sepsis suspicion, using small volumes of sample (25 µL) in a clinically relevant range of concentrations (0.5-150 ng mL-1). The good agreement between PCT levels obtained by our micromotor-based method and routine immunofluorescence hospital determination demonstrates the feasibility for the analysis in VLBWI samples and its potential as a point-of-care diagnostic tool for sepsis.

Magnetic Reduced Graphene Oxide/Nickel/Platinum Nanoparticles Micromotors for Mycotoxin Analysis.

Magnetic reduced graphene oxide/nickel/platinum nanoparticles (rGO/Ni/PtNPs) micromotors for mycotoxin analysis in food samples were developed for food-safety diagnosis. While the utilization of self-propelled micromotors in bioassays has led to a fundamentally new approach, mainly due to the greatly enhanced target-receptor contacts owing to their continuous movement around the sample and the associated mixing effect, herein the magnetic properties of rGO/Ni/PtNPs micromotors for mycotoxin analysis are additionally explored. The micromotor-based strategy for targeted mycotoxin biosensing focused on the accurate control of micromotor-based operations: 1) on-the-move capture of free aptamers by exploiting the adsorption (outer rGO layer) and catalytic (inner PtNPs layer) properties and 2) micromotor stopped flow in just 2 min by exploiting the magnetic properties (intermediate Ni layer). This strategy allowed fumonisin B1 determination with high sensitivity (limit of detection: 0.70 ng mL-1 ) and excellent accuracy (error: 0.05 % in certified reference material and quantitative recoveries of 104±4 % in beer) even in the presence of concurrent ochratoxin A (105-108±8 % in wines). These results confirm the developed approach as an innovative and reliable analytical tool for food-safety monitoring, and confirm the role of micromotors as a new paradigm in analytical chemistry.

Aligned copper nanowires as a cut-and-paste exclusive electrochemical transducer for free-enzyme highly selective quantification of intracellular hydrogen peroxide in cisplatin-treated cells.

The role and reliable quantification of intracellular hydrogen peroxide during cancer therapy constitutes an unexplored and fascinating application. In this work, we report the fabrication of vertically aligned copper nanowires (v-CuNWs) using electrosynthesis on templates, and their application as a cut-and-paste exclusive and flexible electrochemical transducer. This easily adaptable electrodic platform is demonstrated for a fast, simple and free-enzyme selective quantification of intracellular hydrogen peroxide in Cisplatin-treated human renal HK-2 cells. The v-CuNWs sensor was compared with an HRP-enzyme-based biosensor showing excellent correlation and indicates the good selectivity and analytical performance of the v-CuNWs. This sensing approach opens novel avenues for monitoring cell death processes and shows the potential of H2O2 as a cellular damage biomarker, with a clear potency for further developments for in vitro diagnosis and its implication in cancer therapy.

Self-Propelled Enzyme-Based Motors for Smart Mobile Electrochemical and Optical Biosensing.

A millimeter-sized tubular motor for mobile biosensing of H2O2 in environmental and relevant clinical samples is reported. The concept relies on the self-propelled motion by the Marangoni effect, where the asymmetric release of SDS surfactant induces fluid convection and rapid dispersion of horseradish peroxidase (HRP) enzyme into the sample solution. This efficient movement together with the continuous release of fresh enzyme leads to greatly accelerated enzymatic reaction processes without the need of external stirring or chemical and physical attachment of the enzyme as in common classical biosensing approaches. In this strategy, the use of a single millimeter-sized tubular motor during 120 s allows the reliable and accurate quantification of hydrogen peroxide in a set of different matrices such as tap and mineral waters, urine, plasma, and tumor cell cultures treated with antineoplasic Cisplatin without any previous sample preparation. Furthermore, detection can be performed electrochemically, optically, and via visual detection, which makes this approach a clear candidate as a point-of-care analytical tool.

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors.

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.