A dual colorimetric-electrochemical microfluidic paper-based analytical device for point-of-care testing of ischemic strokes.

A novel microfluidic paper-based analytical device with dual colorimetric and electrochemical detection (dual µPAD) was developed for the assessment of transferrin saturation (TSAT) in samples from ischemic stroke patients. TSAT was calculated from the ratio between transferrin-bound iron, which was colorimetrically measured, and the total iron-binding capacity, which was electrochemically measured. To this end, a µPAD was smartly designed, which integrated both colorimetric and electrochemical detection reservoirs, communicating via a microchannel acting as a chemical reactor, and with preloading/storing capabilities (reagent-free device). This approach allowed the dual and simultaneous determination of both parameters, providing an improvement in the reliability of the results due to an independent signal principle and processing. The µPADs were validated by analyzing a certified reference material, showing excellent accuracy (Er ≤ 5%) and precision (RSD ≤ 2%). Then they were applied to the analysis of diagnosed serum samples from ischemic stroke patients. The results were compared to those provided by a free-interference method (urea-PAGE). Impressively, both methods exhibited a good correlation (r = 0.96, p < 0.05) and no significant differences were found between them (slope 1.0 ± 0.1 and the intercept 1 ± 4, p < 0.05), demonstrating the excellent accuracy of our approach during the analysis of complex samples from ischemic stroke patients, using just 90 µL of clinical samples and taking less than 90 min in comparison with the 18 hours required by the urea-PAGE approach. The developed fully integrated colorimetric-electrochemical µPAD is a promising ready to use reagent-free device for the point-of-care testing of TSAT, which can be used to assist physicians in the fast diagnosis and prognosis of ischemic strokes, where the decision-time is crucial for the patient's survival.

Laser-induced 2D/0D graphene-nanoceria freestanding paper-based films for on-site hydrogen peroxide monitoring in no-touch disinfection treatments.

A one-shot CO2 laser-based strategy to generate conductive reduced graphene oxide (rGO) decorated with nanoceria (nCe) is proposed. The 2D/0D rGO-nCe films, integrated as catalytic sensing layers in paper-based sensors, were employed for on-site monitoring of indoor fogging treatments against Listeria monocytogenes (Lm), a ubiquitous pathogenic bacterium. The rGO-nCe laser-assisted synthesis was optimized to preserve the rGO film morphological and electron-transfer features and simultaneously integrate catalytic nCe. The films were characterized by microscopical (SEM), spectroscopical (EDX, Raman, and FTIR), and electrochemical techniques. The most performing film was integrated into a nitrocellulose substrate, and the complete sensor was assembled via a combination of xurography and stencil printing. The rGO-nCe sensor's catalytic activity was proved toward the detection of H2O2, obtaining sensitive determination (LOD = 0.3 µM) and an extended linear range (0.5-1500 µM). Eventually, the rGO-nCe sensor was challenged for the real-time continuous monitoring of hydrogen peroxide aerosol during no-touch fogging treatment conducted following the EU's recommendation for biocidal product use. Treatment effectiveness was proved toward three Lm strains characterized by different origins, i.e., type strain ATCC 7644, clinical strain 338, and food strain 641/6II. The sensor allows for discrimination and quantification treatments at different environmental biocidal amounts and fogging times, and correlates with the microbiological inhibition, promoting the proposed sensor as a useful tool to modulate and monitor no-touch treatments.

Spark-Discharge-Activated 3D-Printed Electrochemical Sensors.

3D printing technology is a tremendously powerful technology to fabricate electrochemical sensing devices. However, current conductive filaments are not aimed at electrochemical applications and therefore require intense activation protocols to unleash a suitable electrochemical performance. Current activation methods based on (electro)chemical activation (using strong alkaline solutions and organic solvents and/or electrochemical treatments) or combined approaches are time-consuming and require hazardous chemicals and dedicated operator intervention. Here, pioneering spark-discharge-activated 3D-printed electrodes were developed and characterized, and it was demonstrated that their electrochemical performance was greatly improved by the effective removal of the thermoplastic support polylactic acid (PLA) as well as the formation of sponge-like and low-dimensional carbon nanostructures. This reagent-free approach consists of a direct, fast, and automatized spark discharge between the 3D-electrode and the respective graphite pencil electrode tip using a high-voltage power supply. Activated electrodes were challenged toward the simultaneous voltammetric determination of dopamine (DP) and serotonin (5-HT) in cell culture media. Spark discharge has been demonstrated as a promising approach for conductive filament activation as it is a fast, green (0.94 GREEnness Metric Approach), and automatized procedure that can be integrated into the 3D printing pipeline.

Molecular Memory Micromotors for Fast Snake Venom Toxin Dynamic Detection.

The analysis and detection of snake venom toxins are a matter of great importance in clinical diagnosis for fast treatment and the discovery of new pharmaceutical products. Current detection methods have high associated costs and require the use of sophisticated bioreceptors, which in some cases are difficult to obtain. Herein, we report the synthesis of template-based molecularly imprinted micromotors for dynamic detection of α-bungarotoxin as a model toxin present in the venom of many-banded krait (Bungarus multicinctus). The specific recognition sites are built-in in the micromotors by incubation of the membrane template with the target toxin, followed by a controlled electrodeposition of a poly(3,4-ethylenedioxythiophene)/poly(sodium 4-styrenesulfonate) polymeric layer, a magnetic Ni layer to promote magnetic guidance and facilitate washing steps, and a Pt layer for autonomous propulsion in the presence of hydrogen peroxide. The enhanced fluid mixing and autonomous propulsion increase the likelihood of interactions with the target analyte as compared with static counterparts, retaining the tetramethylrhodamine-labeled α-bungarotoxin on the micromotor surface with extremely fast dynamic sensor response (after just 20 s navigation) in only 3 µL of water, urine, or serum samples. The sensitivity achieved meets the clinically relevant concentration postsnakebite (from 0.1 to 100 µg/mL), illustrating the feasibility of the approach for practical applications. The selectivity of the protocol is very high, as illustrated by the absence of fluorescence in the micromotor surface in the presence of α-cobratoxin as a representative toxin with a size and structure similar to those of α-bungarotoxin. Recoveries higher than 95% are obtained in the analysis of urine- and serum-fortified samples. The new strategy holds considerable promise for fast, inexpensive, and even onsite detection of several toxins using multiple molecularly imprinted micromotors with tailored recognition abilities.

Gold Nanoparticle-Decorated Catalytic Micromotor-Based Aptassay for Rapid Electrochemical Label-Free Amyloid-ß42 Oligomer Determination in Clinical Samples from Alzheimer's Patients.

Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-ß protein oligomers (AßO) (mainly AßO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aß monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AßO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AßO42 specific thiolated-aptamer (AptAßO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AßO42 (MMGO-AuNPs-AptAßO42-AßO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AßO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 µL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AßO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

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.

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.

Print-Pause-Print Fabrication of Tailored Electrochemical Microfluidic Devices.

Three-dimensional (3D) printing technology has emerged as a powerful technology for the fabrication of low-cost microfluidics. Nevertheless, the fabrication of microfluidic devices integrating high-performance electrochemical sensors in practical applications is still an open challenge. Although automatic fabrication of the microfluidic device and the electrodes can be successfully carried out using a one-step multimaterial fused filament fabrication (FFF) approach, the as-printed electrochemical performance of these electrodes is not good enough for chemical (bio)sensing and their surface modification is challenging because after closing the channel there is no physical access to the electrode. Thus, here a pause-print-pause (PPP) microfabrication approach was implemented. The fabrication was paused before printing the microfluidics, and the filament-based electrodes were directly modified on the printing bed via stencil printing, drop casting, and electrodeposition. To exemplify this versatile workflow, the design of a microfluidic glucose sensor was proposed. To this end, first, the working and counter electrodes were stencil printed with graphite ink while the reference electrode was stencil printed with Ag|AgCl ink. Then, Prussian blue was formed on the working electrode either by drop casting or by electrodeposition, and glucose oxidase was drop cast on top. At this point, the microfabrication process was resumed, and the microfluidics were printed on top of the modified electrodes to complete the construction of hybrid electrochemical fluidic fused filament fabricated devices (h-eF4Ds). This print-pause-print approach is not limited to ink-based electrodes or glucose oxidase, and we envisage these results will pave the way for the effective integration of electrodes in microfluidic devices in a simple and clean-room-free approach, allowing the development of highly customized eF4Ds for a plethora of analytes with high significance.

Photoactive Au@MoS2 Micromotors for Dynamic Surface-Enhanced Raman Spectroscopy Sensing.

Photophoretic Au@MoS2 micromotors are used as smart mobile substrates for dynamic surface-enhanced Raman spectroscopy (SERS) sensing. The photophoretic capabilities and swarming-like propulsion of the micromotors allow for their schooling and accumulation in the measuring spot, increasing the density of SERS-active gold nanoparticles for Raman mapping and, simultaneously, the preconcentration of the target analyte. The generation of "hot-microflake spots" directly in the Raman irradiation point results in a 15-18-fold enhancement in the detection of crystal violet without the requirement for additional external sources for propulsion. Moreover, the reproducible collective micromotor motion does not depend on the exact position of the laser spot concerning individual micromotors, which greatly simplifies the experimental setup, avoiding the requirements of sophisticated equipment. The strategy was further applied for the detection of malachite green and paraquat with a good signal enhancement. The new on-the-move-based SERS strategy holds great promise for on-site detection with portable instrumentation in a myriad of environmental monitoring and clinical applications.

The rise of metal-organic framework based micromotors.

Micromotors (MMs) are micro and nanoscale devices capable of converting energy into autonomous motion. Metal-organic frameworks (MOFs) are crystalline materials that display exceptional properties such as high porosity, internal surface areas, and high biocompatibility. As such, MOFs have been used as active materials or building blocks for MMs. In this highlight, we describe the evolution of MOF-based MMs, focusing on the last 3 years. First, we covered the main propulsion mechanisms and designs, from catalytic to fuel-free MOF-based MMs. Secondly, we discuss recent applications of new fuel-free MOFs MM to give a critical overview of the current challenges of this blooming research field. The advantages and challenges discussed provide a useful guide for the design of the next generation MOF MMs toward real-world applications.

Paper-Based Analytical Devices for Accurate Assessment of Transferrin Saturation in Diagnosed Clinical Samples from Ischemic Stroke Patients.

For the first time, a paper-based analytical device (PAD) was developed for the assessment of transferrin saturation (TSAT), which is defined as the ratio between iron bound to transferrin (Tf) and the total iron-binding capacity (TIBC) of Tf. Both parameters were simultaneously measured on the same PAD using ferrozine as a chromophore and a smartphone as the color reader. To this end, Tf was first isolated from serum using anti-Tf immunomagnetic beads to ensure that only the Tf-bound iron was measured, improving the selectivity and accuracy of TSAT assessment. To demonstrate the practical utility of the device, it was validated by analyzing a certified reference material, showing excellent accuracy (Er < 4%) and good precision (RSD ≤ 6%). Finally, 18 diagnosed serum samples from ischemic stroke patients were analyzed by this approach, and the results were compared with those obtained by urea-PAGE, showing not only an excellent correlation (r = 0.93, p < 0.05) but that the PAD approach has become statistically identical to the free-interference urea-PAGE. In comparison with the slow, tedious, and non-miniaturized-PAGE, this PAD approach exhibited attractive characteristics such as low cost, disposability, and connectivity, showing great potential for future point-of-care testing, especially in developing countries and/or remote areas, where access to medical or clinical facilities is limited.

Photoresponsive MoS2 and WS2 microflakes as mobile biocide agents.

A fuel-free strategy for the eradication of Escherichia coli and Staphylococcus aureus biofilms using WS2 and MoS2 photophoretic microflakes is described. The microflakes were prepared by liquid-phase exfoliation of the materials. Under electromagnetic irradiation at 480 or 535 nm, the microflakes experience a fast collective behavior at speeds of over 300 µm s-1 due to photophoresis. Simultaneously to their motion, reactive oxygen species are generated. The fast microflake schooling into multiple moving swarms results in a highly efficient "collision" platform that disrupts the biofilm, enhancing radical oxygen species' contact with the bacteria for their inactivation. As such, removal biofilm mass rates of over 90% and 65% are achieved using the MoS2 and WS2 microflakes in the treatment of Gram-negative E. coli and Gram-positive S. aureus biofilms after 20 min. Much lower removal biofilm mass rates (30%) are obtained under static conditions, revealing the crucial role of microflake movement and radical generation in the active eradication of biofilms. Much higher removal efficiencies are observed in biofilm deactivation as compared with the use of free antibiotics, which are not able to destroy the densely packed biofilms. The new moving microflakes hold considerable promise for the treatment of antibiotic-resistant bacteria.

Analysis of microsamples by miniaturized magnetic-based pipette tip microextraction: determination of free cortisol in serum and urine from very low birth weight preterm newborns.

Miniaturized magnetic-based pipette tip microextraction is presented as a sample preparation approach for microsamples. It involves quick dispersion of a diminutive amount of a magnetic sorbent material in a low-volume sample (10 µL) to entrap the target analytes. Next, the dispersion is aspirated using a (semi)automatic pipette through a pipette tip with a small cubic neodymium magnet inside, which retrieves the magnetic sorbent containing the analytes. After discarding the rest of the sample, the sorbent is properly rinsed by aspirating/dispensing deionized water, and then, the analytes are eluted by aspirating/dispensing an appropriate solvent. This approach was employed for the determination of free cortisol in serum and urine from very low birth weight preterm newborns, a vulnerable patient group who present low availability for sampling biological fluids. A magnetic immunosorbent made of a cortisol antibody was employed for the selective extraction, followed by liquid chromatography-tandem mass spectrometry. Good analytical features were obtained, such as limits of detection and quantification of 0.08 and 0.27 ng mL-1, respectively, linearity up to 50 ng mL-1 (R2 > 0.999), RSD values under 15% and relative recoveries between 91 and 111%. The cross-reactivity with other glucocorticoids (i.e., cortisone and prednisolone) was evaluated to show the selectivity of the extraction. Finally, the method applicability was demonstrated towards the determination of free cortisol in the serum and urine samples from low birth weight preterm newborns.

Integrated calibration and serum iron in situ analysis into an array microfluidic paper-based analytical device with smartphone readout.

In this work, a colorimetric microfluidic paper-based analytical device (µPAD) combined with a smartphone readout was proposed for the determination of serum iron (Fe3+), which is linked to transferrin. Firstly, Fe3+ was selectively isolated and preconcentrated from serum by using anti-transferrin immunomagnetic beads (anti-Tf-MBs). Secondly, Fe3+ is reduced to Fe2+ by a hydroxylamine solution (pH 4.8) and then measured in the µPAD, which contains the colorimetric reagent ferrozine. Finally, the intensity of the purple color formed in the µPAD was measured by a smartphone. The approach exhibited an excellent linear correlation (r = 0.996) and good limit of detection (0.3 µg mL-1). Moreover, a certified reference material (human serum) was analyzed by this approach, showing an excellent accuracy (Er < 4%) and inter-device reproducibility (RSD = 1%, n = 3). Interestingly, the µPAD array-design allowed the simultaneous analysis of different samples, improving the sample throughput (up to 5 samples in 130 min, using 100 µL each), and the integration of calibration and analysis into the same device, simplifying the analysis without losing accuracy or sensitivity, and avoiding inter-device variability, which constituted an added value to this approach. These disposable µPADs meet several requirements of point-of-care testing (POCT) because it is cheap, portable, easy-to-use, sensitive, and specific. Therefore, it may be an interesting way for measuring patients' serum iron levels in situ with reliability, especially, in developing countries, where the prevalence of iron deficiency and iron-deficiency anemia is higher and there are lower health resources than in developed countries.

Nanomaterials meet surface-enhanced Raman scattering towards enhanced clinical diagnosis: a review.

Surface-enhanced Raman scattering (SERS) is a very promising tool for the direct detection of biomarkers for the diagnosis of i.e., cancer and pathogens. Yet, current SERS strategies are hampered by non-specific interactions with co-existing substances in the biological matrices and the difficulties of obtaining molecular fingerprint information from the complex vibrational spectrum. Raman signal enhancement is necessary, along with convenient surface modification and machine-based learning to address the former issues. This review aims to describe recent advances and prospects in SERS-based approaches for cancer and pathogens diagnosis. First, direct SERS strategies for key biomarker sensing, including the use of substrates such as plasmonic, semiconductor structures, and 3D order nanostructures for signal enhancement will be discussed. Secondly, we will illustrate recent advances for indirect diagnosis using active nanomaterials, Raman reporters, and specific capture elements as SERS tags. Thirdly, critical challenges for translating the potential of the SERS sensing techniques into clinical applications via machine learning and portable instrumentation will be described. The unique nature and integrated sensing capabilities of SERS provide great promise for early cancer diagnosis or fast pathogens detection, reducing sanitary costs but most importantly allowing disease prevention and decreasing mortality rates.

Digital manufacturing for accelerating organ-on-a-chip dissemination and electrochemical biosensing integration.

Organ on-a-chip (OoC) is a promising technology that aims to recapitulate human body pathophysiology in a more precise way to advance in drug development and complex disease understanding. However, the presence of OoC in biological laboratories is still limited and mainly restricted to laboratories with access to cleanroom facilities. Besides, the current analytical methods employed to extract information from the organ models are endpoint and post facto assays which makes it difficult to ensure that during the biological experiment the cell microenvironment, cellular functionality and behaviour are controlled. Hence, the integration of real-time biosensors is highly needed and requested by the OoC end-user community to provide insight into organ function and responses to stimuli. In this context, electrochemical sensors stand out due to their advantageous features like miniaturization capabilities, ease of use, automatization and high sensitivity and selectivity. Electrochemical sensors have been already successfully miniaturized and employed in other fields such as wearables and point-of-care devices. We have identified that the explanation for this issue may be, to a large extent, the accessibility to microfabrication technologies. These fields employ preferably digital manufacturing (DM), which is a more accessible microfabrication approach regardless of funding and facilities. Therefore, we envision that a paradigm shift in microfabrication that adopts DM instead of the dominating soft lithography for the in-lab microfabrication of OoC devices will contribute to the dissemination of the field and integration of the promising real-time sensing.

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.