Intradermal Lactate Monitoring Based on a Microneedle Sensor Patch for Enhanced In Vivo Accuracy.

Lactate is an important diagnostic and prognostic biomarker of several human pathological conditions, such as sepsis, malaria, and dengue fever. Unfortunately, due to the lack of reliable analytical decentralized platforms, the determination of lactate yet relies on discrete blood-based assays, which are invasive and inefficient and may cause tension and pain in the patient. Herein, we demonstrate the potential of a fully integrated microneedle (MN) sensing system for the minimally invasive transdermal detection of lactate in an interstitial fluid (ISF). The originality of this analytical technology relies on: (i) a strategy to provide a uniform coating of a doped polymer-based membrane as a diffusion-limiting layer on the MN structure, optimized to perform full-range lactate detection in the ISF (linear range of response: 0.25-35 mM, 30 s assay time, 8 h operation), (ii) double validation of ex vivo and in vivo results based on ISF and blood measurements in rats, (iii) monitoring of lactate level fluctuations under the administration of anesthesia to mimic bedside clinical scenarios, and (iv) in-house design and fabrication of a fully integrated and portable sensing device in the form of a wearable patch including a custom application and user-friendly interface in a smartphone for the rapid, routine, continuous, and real-time lactate monitoring. The main analytical merits of the lactate MN sensor include appropriate selectivity, reversibility, stability, and durability by using a two-electrode amperometric readout. The ex-vivo testing of the MN patch of preconditioned rat skin pieces and euthanized rats successfully demonstrated the accuracy in measuring lactate levels. The in vivo measurements suggested the existence of a positive correlation between ISF and blood lactate when a lag time of 10 min is considered (Pearson's coefficient = 0.85, mean difference = 0.08 mM). The developed MN-based platform offers distinct advantages over noncontinuous blood sampling in a wide range of contexts, especially where access to laboratory services is limited or blood sampling is not suitable. Implementation of the wearable patch in healthcare could envision personalized medicine in a variety of clinical settings.

Demonstrating the Analytical Potential of a Wearable Microneedle-Based Device for Intradermal CO2 Detection.

Monitoring of carbon dioxide (CO2) body levels is crucial under several clinical conditions (e.g., human intensive care and acid-base disorders). To date, painful and risky arterial blood punctures have been performed to obtain discrete CO2 measurements needed in clinical setups. Although noninvasive alternatives have been proposed to assess CO2, these are currently limited to benchtop devices, requiring trained personnel, being tedious, and providing punctual information, among other disadvantages. To the best of our knowledge, the literature and market lack a wearable device for real-time, on-body monitoring of CO2. Accordingly, we have developed a microneedle (MN)-based sensor array, labeled as CO2-MN, comprising a combination of potentiometric pH- and carbonate (CO32-)-selective electrodes together with the reference electrode. The CO2-MN is built on an epidermal patch that allows it to reach the stratum corneum of the skin, measuring pH and CO32- concentrations directly into the interstitial fluid (ISF). The levels for the pH-CO32- tandem are then used to estimate the PCO2 in the ISF. Assessing the response of each individual MN, we found adequate response time (t95 < 5s), sensitivity (50.4 and -24.6 mV dec-1 for pH and CO32-, respectively), and stability (1.6 mV h-1 for pH and 2.1 mV h-1 for CO32-). We validated the intradermal measurements of CO2 at the ex vivo level, using pieces of rat skin, and then, with in vivo assays in anesthetized rats, showing the suitability of the CO2-MN wearable device for on-body measurements. A good correlation between ISF and blood CO2 concentrations was observed, demonstrating the high potential of the developed MN sensing technology as an alternative to blood-based analysis in the near future. Moreover, these results open new horizons in the noninvasive, real-time monitoring of CO2 as well as other clinically relevant gases.

Fully Integrated Wearable Device for Continuous Sweat Lactate Monitoring in Sports.

The chemical digitalization of sweat using wearable sensing interfaces is an attractive alternative to traditional blood-based protocols in sports. Although sweat lactate has been claimed to be a relevant biomarker in sports, an analytically validated wearable system to prove that has not yet been developed. We present a fully integrated sweat lactate sensing system applicable to in situ perspiration analysis. The device can be conveniently worn in the skin to monitor real-time sweat lactate during sports, such as cycling and kayaking. The novelty of the system is threefold: advanced microfluidics design for sweat collection and analysis, an analytically validated lactate biosensor based on a rational design of an outer diffusion-limiting membrane, and an integrated circuit for signal processing with a custom smartphone application. The sensor covering the range expected for lactate in sweat (1-20 mM), with appropriate sensitivity (-12.5 ± 0.53 nA mM-1), shows an acceptable response time (<90 s), and the influence of changes in pH, temperature, and flow rate are neglectable. Also, the sensor is analytically suitable with regard to reversibility, resilience, and reproducibility. The sensing device is validated through a relatively high number of on-body tests performed with elite athletes cycling and kayaking in controlled environments. Correlation outcomes between sweat lactate and other physiological indicators typically accessible in sports laboratories (blood lactate, perceived exhaustion, heart rate, blood glucose, respiratory quotient) are also presented and discussed in relation to the sport performance monitoring capability of continuous sweat lactate.

In Vivo Transdermal Multi-Ion Monitoring with a Potentiometric Microneedle-Based Sensor Patch.

Microneedle sensor technology offers exciting opportunities for decentralized clinical analyses. A novel issue puts forward herein is to demonstrate the uniqueness of membrane-based microneedles to accomplish real-time, on-body monitoring of multiple ions simultaneously. The use of multi-ion detection is clinically relevant since it is expected to provide a more complete and reliable assessment of the clinical status of a subject concerning electrolyte disorders and others. We present a microneedle system for transdermal multiplexed tracing of pH, Na+, K+, Ca2+, Li+, and Cl-. The device consists of an array of seven solid microneedles externally modified to provide six indicator electrodes, each selective for a different ion, and a common reference electrode, all integrated into a wearable patch read in a potentiometric mode. We show in vitro measurements at the expected clinical levels, resulting in a fast response time, excellent reversibility and repeatability, and adequate selectivity. Close-to-Nernstian sensitivity, sufficient stability and resiliency to skin penetration guarantee the sensor's success in transdermal measurements, which we demonstrate through ex vivo (with pieces of rat skin) and in vivo (on-body measurements in rats) tests. Accuracy is evaluated by comparison with gold standard techniques to characterize collected dermal fluid, blood, and serum. In the past, interstitial fluid (ISF) analysis has been challenging due to difficult sample collection and analysis. For ions, this has resulted in extrapolations from blood concentrations (invasive tests) rather than pure measurements in ISF. The developed microneedle patch is a relevant analytical tool to address this information gap.

Intradermal Glycine Detection with a Wearable Microneedle Biosensor: The First In Vivo Assay.

Glycine (GLY) is gaining importance in medical diagnoses due to its relationship with multiple physiological functions. Today, GLY is exclusively analyzed using instrumentation centralized in clinical labs, and a tangible point-of-care tool that gathers real-time data from the patient for effective and fast evaluations is lacking. Relevant clinical advances are expected as soon as the rapid provision of both punctual and continuous measurements is possible. In that context, this work presents a microneedle (MN)-based biosensor for intradermal GLY detection in interstitial fluid (ISF). The MN tip is externally tailored to detect GLY levels through the hydrogen peroxide formed in its reaction with a quinoprotein-based GLY oxidase enzyme. The analytical performance of the MN biosensor indicates a fast response time (<7 s); acceptable reversibility, reproducibility, and stability; as well as a wide linear range of response (25-600 µM) that covers the physiological levels of GLY in ISF. The MN biosensor conveniently exhibits high selectivity for GLY over other compounds commonly found in ISF, and the response is not influenced by temperature, pH, or skin insertions. Validated intradermal measurements of GLY were obtained at the in vitro (with pieces of rat skin), ex vivo (on-body tests of euthanized rats) and in vivo (on-body tests of anesthetized rats) levels, demonstrating its ability to produce accurate physiological data. The developed GLY MN biosensor is skin-wearable and provides reliable, real-time intradermal GLY measurements in ISF by means of a minimally invasive approach.

Prussian Blue/Chitosan Micromotors with Intrinsic Enzyme-like Activity for (bio)-Sensing Assays.

Prussian Blue (PB)/chitosan enzyme mimetic tubular micromotors are used here for on-the-fly (bio)-sensing assays. The micromotors are easily prepared by direct deposition of chitosan into the pores of a membrane template and in situ PB synthesis during hydrogel deposition. Under judicious pH control, PB micromotors display enzyme mimetic capabilities with three key functions on board: the autonomous oxygen bubble propulsion (with PB acting as a catalase mimic for hydrogen peroxide decomposition), 3,3',5,5'-tetramethylbenzidine (TMB) oxidation (with PB acting as a peroxidase mimic for analyte detection), and as a magnetic material (to simplify the (bio)-sensing steps). In connection with chitosan capabilities, these unique enzyme mimetic micromotors are further functionalized with acetylthiocholinesterase enzyme (ATChE) to be explored in fast inhibition assays (20 min) for the colorimetric determination of the nerve agent neostigmine, with excellent analytical performance in terms of quantification limit (0.30 µM) and concentration linear range (up to 500 µM), without compromising efficient micromotor propulsion. The new concept illustrated holds considerable potential for a myriad of (bio)-sensing applications, including forensics, where this conceptual approach remains to be explored. Micromotor-based tests to be used in crime scenes are also envisioned due to the reliable neostigmine determination in unpretreated samples.

An on-chip microfluidic-based electrochemical magneto-immunoassay for the determination of procalcitonin in plasma obtained from sepsis diagnosed preterm neonates.

A novel on-chip electrochemical magneto-immunoassay for the determination of procalcitonin (PCT) has been proposed. The strategy involved the on-line performing of the biorecognition event and detection on the thin-film microfluidic gold electrode chamber operating at E = -0.20 V (vs. Au). The complete assay was performed in less than 15 minutes using only 25 µL of the sample, covering the entire range of clinically relevant PCT concentrations in sepsis diagnosis with a limit of detection and quantification of 0.02 ng mL-1 and 0.05 ng mL-1, respectively (the sepsis diagnosis threshold: 0.5 ng mL-1). The on-chip electrochemical magneto-immunoassay provided excellent results in the analysis of very unique samples obtained from preterm neonates admitted with suspected sepsis, in which the sample volume is hardly available. These characteristics fulfill the POCT requirements for PCT determination in the whole clinically relevant concentration range. Because of the high clinical relevance and the important role of PCT in sepsis, this approach opens new perspectives for sepsis diagnosis and therapy guidance using low volume samples.

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.

On-the-fly rapid immunoassay for neonatal sepsis diagnosis: C-reactive protein accurate determination using magnetic graphene-based micromotors.

Based on the exceptional and new opened biosensing possibilities of self-propelled micromotors, a micromotor-based immunoassay (MIm) has smartly been designed for C-reactive protein (CRP) determination in plasma of preterm infants with sepsis suspicion. The design of the micromotors involved the electrosynthesis of a carbon-based outer layer (for antibody functionalization), an intermediate Ni layer (for magnetic guidance and stopped flow operations) and PtNPs inner catalytic layer (for catalytic bubble propulsion). Micromotors biofunctionalization on the outer layer (using carbon black (CB), reduced graphene oxide (rGO) and multi-walled carbon nanotubes (MWCNTs), and biocompatible propulsion capabilities, were carefully studied. Magnetic rGO/Ni/PtNPs micromotors exhibited the most efficient and reproducible (CV = 9%) anti-CRP functionalization, controlled stopped-flow operations as well as efficient bubble propulsion (1% H2O2, 1,5% NaCh, speed 140 µm s-1). Analytical performance of MIm was excellent, allowing the direct (without dilution), sensitive (LOD = 0.80 µg/mL), and accurate CRP determination (Er = 1%) in hardly available preterm babies' plasma samples with suspected sepsis using very low volumes (<10 µL) and in just 5 min of on-the-fly bioassay. Overall, the results obtained allowed the fast and reliable sepsis diagnostics in preterm babies' individuals with suspected sepsis, not only proving the usefulness of the approach as its potential utilization as point-of-care device for clinical analysis but drawing new horizons in extremely low sample volumes-based diagnostics.

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.

Electrochemical Microfluidic Micromotors-Based Immunoassay for C-Reactive Protein Determination in Preterm Neonatal Samples with Sepsis Suspicion.

Online coupling of a micromotor-based immunoassay and a microfluidic electrochemical detection was explored as a new approach for C-reactive protein (CRP) determination in serum and preterm neonatal plasma samples with sepsis suspicion. The approach combines the advantages of micromotors (self-fluid mixing capabilities leading to a faster assay in low sample volumes) and electrochemical microfluidic (flow-controlled ultraminiaturized electrochemical detection, high sensitivity, and low-cost) technologies. Both technologies elegantly meet the point of care testing or bed side device requirements such as low analysis times, miniaturization and simplification, and single use. Anti-CRP functionalized micromotors (anti-CRP-rGO(reduced graphene oxide)/Ni/PtNPs (platinum nanoparticles))-based immunoassay coupled to thin layer Au-based electrochemical microfluidics operating at -0.20 V under controlled fluidic detection operations (30 µL min-1) allowed the sensitive (LOD = 0.54 µg/mL) and accurate CRP determination using very low volume preterm neonatal clinical samples (<10 µL) in just 8 min of total assay time. These excellent analytical characteristics obtained linked to the full automatization of the immunoassay allowed the fast and accurate determination of CRP in hardly available clinical samples as those coming from preterm infants with suspected sepsis. These results demonstrated the usefulness of the approach which meets the clinical requirements as a future point-of-care device for clinical analysis.

Toward Early Diagnosis of Late-Onset Sepsis in Preterm Neonates: Dual Magnetoimmunosensor for Simultaneous Procalcitonin and C-Reactive Protein Determination in Diagnosed Clinical Samples.

Early diagnosis of sepsis, combining blood cultures and inflammation biomarkers, continues to be a challenge, especially in very low birth weight (VLBW) infants because of limited availability of blood samples. Traditional diagnostic procedures are cumbersome, not fast enough, and require relatively large volumes of sample. Empiric use of antibiotics, before diagnostic confirmation, is required to decrease mortality, leading to potential antibiotic resistance and side effects in VLBW infants. To solve such a serious problem, a dual magnetoimmunosensor is proposed for simultaneous assessment of two of the most important sepsis biomarkers: procalcitonin (PCT for early phase) and C-reactive protein (CRP for late phase). This "sample-to-result" approach exhibited excellent sensitivity, selectivity, precision, and stability using low sample volumes (<30 µL) and under 20 min of total assay. The analytical usefulness of the approach was demonstrated by analyzing clinically relevant samples of preterm neonates with suspicion of 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.

Biosensing Strategy for Simultaneous and Accurate Quantitative Analysis of Mycotoxins in Food Samples Using Unmodified Graphene Micromotors.

A high-performance graphene-based micromotor strategy for simultaneous, fast, and reliable assessment of two highly concerning mycotoxins (fumonisin B1 (FB1) and ocratoxin A (OTA)) has successfully been developed. The assay principle is based on the selective recognition from aptamers to the target mycotoxins and further "on-the-move" fluorescence quenching of the free aptamer in the outer layer of unmodified reduced graphene (rGO; sensing layer) micromotors. Template-prepared rGO/platinum nanoparticles (PtNPs) tubular micromotors were synthesized rapidly and inexpensively by the direct electrodeposition within the conical pores of a polycarbonate template membrane. The new wash-free approach offers using just 1 µL of sample, a simultaneous and rapid "on-the-fly" detection (2 min) with high sensitivity (limits of detection of 7 and 0.4 ng/mL for OTA and FB1, respectively), and high selectivity. Remarkable accuracy (Er < 5%) during the mycotoxin determination in certified reference material as well as excellent quantitative recoveries (96-98%) during the analysis of food samples were also obtained. The excellent results obtained allow envisioning an exciting future for the development of novel applications of catalytic micromotors in unexplored fields such as food safety diagnosis.