A reagentless molecularly imprinted polymer-based electrochemical biosensor for single-step detection of troponin I in biofluids.

Reagentless molecular-imprinted polymer (MIP) electrochemical biosensors can offer the next generation of biosensing platforms for the detection of biomarkers owing to their simplicity, cost-efficacy, tunability, robustness, and accuracy. In this work, a novel combination of Prussian blue (PB), coated as an embedded redox probe on a gold working electrode (GWE), and a signal-off MIP assay has been proposed in an electrochemical format for the detection of troponin I (TnI) in biofluids. TnI is a variant exclusive to heart muscles, and its elevated level in the bloodstream is indicative of acute myocardial infarction (AMI). The proposed lab-manufactured PB/MIP electrochemical biosensor, consisting of a simple signal-off MIP assay and a PB redox probe embedded on the GWE surface, is the first of its kind that allows for reagentless, label-free, and single-step electrochemical biosensing of proteins. The preparation steps of the biosensor were fully characterized by cyclic voltammetry (CV), atomic force microscopy (AFM), and Raman spectroscopy. Finally, the performance of the optimized biosensor was investigated through the determination of various concentrations of TnI, ranging from 10 to 100 pg mL-1 within 5 min, in serum and plasma with limits of detection less than 3.6 pg mL-1, and evaluation of selectivity towards TnI using some relevant proteins that exist in biofluids with higher concentrations.

Surface-Based Multimeric Aptamer Generation and Bio-Functionalization for Electrochemical Biosensing Applications.

Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.

Utilizing Gradient Porous Graphene Substrate as the Solid-Contact Layer To Enhance Wearable Electrochemical Sweat Sensor Sensitivity.

Wearable sweat monitoring represents an attractive opportunity for personalized healthcare and for evaluating sports performance. One of the limitations with such monitoring, however, is water layer formation upon cycling of ion-selective sensors, leading to degraded sensitivity and long-term instability. Our report is the first to use chemical vapor deposition-grown, three-dimensional, graphene-based, gradient porous electrodes to minimize such water layer formation. The proposed design reduces the ion diffusion path within the polymeric ion-selective membrane and enhances the electroactive surface for highly sensitive, real-time detection of Na+ ions in human sweat with high selectivity. We obtained a 7-fold enhancement in electroactive surface against 2D electrodes (e.g., carbon, gold), yielding a sensitivity of 65.1 ± 0.25 mV decade-1 (n = 3, RSD = 0.39%), the highest to date for wearable Na+ sweat sensors. The on-body sweat sensing performance is comparable to that of ICP-MS, suggesting its feasibility for health evaluation through sweat.

Nanofluidics for Simultaneous Size and Charge Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane structures that circulate in body fluids and show considerable potential for noninvasive diagnosis. EVs possess surface chemistries and encapsulated molecular cargo that reflect the physiological state of cells from which they originate, including the presence of disease. In order to fully harness the diagnostic potential of EVs, there is a critical need for technologies that can profile large EV populations without sacrificing single EV level detail by averaging over multiple EVs. Here we use a nanofluidic device with tunable confinement to trap EVs in a free-energy landscape that modulates vesicle dynamics in a manner dependent on EV size and charge. As proof-of-principle, we perform size and charge profiling of a population of EVs extracted from human glioblastoma astrocytoma (U373) and normal human astrocytoma (NHA) cell lines.

Are plasmonic optical biosensors ready for use in point-of-need applications?

Plasmonics has drawn significant attention in the area of biosensors for decades due to the unique optical properties of plasmonic resonant nanostructures. While the sensitivity and specificity of molecular detection relies significantly on the resonance conditions, significant attention has been dedicated to the design, fabrication, and optimization of plasmonic substrates. The adequate choice of materials, structures, and functionality goes hand in hand with a fundamental understanding of plasmonics to enable the development of practical biosensors that can be deployed in real life situations. Here we provide a brief review of plasmonic biosensors detailing most recent developments and applications. Besides metals, novel plasmonic materials such as graphene are highlighted. Sensors based on Surface Plasmon Resonance (SPR), Localized Surface Plasmon Resonance (LSPR), and Surface Enhanced Raman Spectroscopy (SERS) are presented and classified based on their materials and structure. In addition, most recent applications to environment monitoring, health diagnosis, and food safety are presented. Potential problems related to the implementation in such applications are discussed and an outlook is presented.

Microscale reactor embedded with Graphene/hierarchical gold nanostructures for electrochemical sensing: application to the determination of dopamine.

An integrated electrochemical sensing platform is presented, in which stable graphene nanosheets are entrapped within hierarchical gold nano/micro islands (NMI) for the selective detection of dopamine. The fabrication method, which combines lithography, electrodeposition and liquid exfoliation, results in a microscale fluidic reactor capable of handling small volumes (10 µl) of sample. This configuration has advantageous properties, including enhanced sensitivity towards current responses from redox reaction of dopamine to dopamine orthoquinone. The NMIs'spatial orientation inhibits the agglomeration of graphene, while their nanostructured interface enhances adhesion to graphene nanosheets. In turn, this leads to an enlarged surface and to an accumulation of free electrons on the electrode surface. The superior electrocatalytic activity for dopamine is attributed to the high density of π-electrons on graphene nanosheets. In addition, the selectivity of the assay in the presence of other interferents is assumed to be a result of the sp2 π-interactions between the negatively charged graphene layer and the aromatic rings of dopamine. At a working potential of 0.15 V vs Ag/AgCl, the assay has a detection limit of 1.13 nM, a linear range of 1 nM- 100 µM, and apparent recoveries of 106% in spiked synthetic urine. Graphical abstractSchematic presentation of an integrated electrochemical sensing platform, in which stable graphene nanosheets are entrapped within hierarchical gold nano/micro islands (NMI) for selective detection of dopamine. Platinum (Pt) wire and Silver/Silver-Chloride (Ag/AgCl) were used as counter and reference electrode, respectively.

Peptide-Mediated Electrochemical Steric Hindrance Assay for One-Step Detection of HIV Antibodies.

Diagnosis of infectious disease in patients, including human immunodeficiency virus (HIV) infection, can be achieved through the detection of specific antibodies produced by the immune system. We have previously shown that macromolecules such as antibodies can be efficiently detected in complex biological samples by sterically inhibiting the hybridization of conjugated complementary DNA strands to electrode-bound DNA strands. Here, we report a peptide-mediated electrochemical steric hindrance hybridization assay, PeSHHA, specially for the detection of antibodies against the gp41 protein of HIV-1. We show that the sensitivity of this PeSHHA can be significantly enhanced using nanostructured electrodes and demonstrate the rapid, one-step detection of HIV-1 antibodies directly in clinical samples.

A review on recent advancements in electrochemical biosensing using carbonaceous nanomaterials.

This review, with 201 references, describes the recent advancement in the application of carbonaceous nanomaterials as highly conductive platforms in electrochemical biosensing. The electrochemical biosensing is described in introduction by classifying biosensors into catalytic-based and affinity-based biosensors and statistically demonstrates the most recent published works in each category. The introduction is followed by sections on electrochemical biosensors configurations and common carbonaceous nanomaterials applied in electrochemical biosensing, including graphene and its derivatives, carbon nanotubes, mesoporous carbon, carbon nanofibers and carbon nanospheres. In the following sections, carbonaceous catalytic-based and affinity-based biosensors are discussed in detail. In the category of catalytic-based biosensors, a comparison between enzymatic biosensors and non-enzymatic electrochemical sensors is carried out. Regarding the affinity-based biosensors, scholarly articles related to biological elements such as antibodies, deoxyribonucleic acids (DNAs) and aptamers are discussed in separate sections. The last section discusses recent advancements in carbonaceous screen-printed electrodes as a growing field in electrochemical biosensing. Tables are presented that give an overview on the diversity of analytes, type of materials and the sensors performance. Ultimately, general considerations, challenges and future perspectives in this field of science are discussed. Recent findings suggest that interests towards 2D nanostructured electrodes based on graphene and its derivatives are still growing in the field of electrochemical biosensing. That is because of their exceptional electrical conductivity, active surface area and more convenient production methods compared to carbon nanotubes. Graphical abstract Schematic representation of carbonaceous nanomaterials used in electrochemical biosensing. The content is classified into non-enzymatic sensors and affinity/ catalytic biosensors. Recent publications are tabulated and compared, considering materials, target, limit of detection and linear range of detection.

Electrochemical sensors and biosensors based on the use of polyaniline and its nanocomposites: a review on recent advances.

Polyaniline and its composites with nanoparticles have been widely used in electrochemical sensor and biosensors due to their attractive properties and the option of tuning them by proper choice of materials. The review (with 191 references) describes the progress made in the recent years in polyaniline-based biosensors and their applications in clinical sensing, food quality control, and environmental monitoring. A first section summarizes the features of using polyaniline in biosensing systems. A subsequent section covers sensors for clinical applications (with subsections on the detection of cancer cells and bacteria, and sensing of glucose, uric acid, and cholesterol). Further sections discuss sensors for use in the food industry (such as for sulfite, phenolic compounds, acrylamide), and in environmental monitoring (mainly pesticides and heavy metal ions). A concluding section summarizes the current state, highlights some of the challenges currently compromising performance in biosensors and nanobiosensors, and discusses potential future directions. Graphical abstract Schematic presentation of electrochemical sensor and biosensors applications based on polyaniline/nanoparticles in various fields of human life including medicine, food industry, and environmental monitoring. The simultaneous use of suitable properties polyaniline and nanoparticles can provide the fabrication of sensing systems with high sensitivity, short response time, high signal/noise ratio, low detection limit, and wide linear range by improving conductivity and the large surface area for biomolecules immobilization.

A Hierarchical 3D Nanostructured Microfluidic Device for Sensitive Detection of Pathogenic Bacteria.

Efficient capture and rapid detection of pathogenic bacteria from body fluids lead to early diagnostics of bacterial infections and significantly enhance the survival rate. We propose a universal nano/microfluidic device integrated with a 3D nanostructured detection platform for sensitive and quantifiable detection of pathogenic bacteria. Surface characterization of the nanostructured detection platform confirms a uniform distribution of hierarchical 3D nano-/microisland (NMI) structures with spatial orientation and nanorough protrusions. The hierarchical 3D NMI is the unique characteristic of the integrated device, which enables enhanced capture and quantifiable detection of bacteria via both a probe-free and immunoaffinity detection method. As a proof of principle, we demonstrate probe-free capture of pathogenic Escherichia coli (E. coli) and immunocapture of methicillin-resistant-Staphylococcus aureus (MRSA). Our device demonstrates a linear range between 50 and 104 CFU mL-1 , with average efficiency of 93% and 85% for probe-free detection of E. coli and immunoaffinity detection of MRSA, respectively. It is successfully demonstrated that the spatial orientation of 3D NMIs contributes in quantifiable detection of fluorescently labeled bacteria, while the nanorough protrusions contribute in probe-free capture of bacteria. The ease of fabrication, integration, and implementation can inspire future point-of-care devices based on nanomaterial interfaces for sensitive and high-throughput optical detection.

Convex lens-induced nanoscale templating.

We demonstrate a new platform, convex lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a convex lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub-30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.

Mixed confinement regimes during equilibrium confinement spectroscopy of DNA.

We have used a combination of fluorescence microscopy experiments and Pruned Enriched Rosenbluth Method simulations of a discrete wormlike chain model to measure the mean extension and the variance in the mean extension of λ-DNA in 100 nm deep nanochannels with widths ranging from 100 nm to 1000 nm in discrete 100 nm steps. The mean extension is only weakly affected by the channel aspect ratio. In contrast, the fluctuations of the chain extension qualitatively differ between rectangular channels and square channels with the same cross-sectional area, owing to the "mixing" of different confinement regimes in the rectangular channels. The agreement between experiment and simulation is very good, using the extension due to intercalation as the only adjustable parameter.

Mimicking nature to develop halide perovskite semiconductors from proteins and metal carbonates.

Halide perovskite (HPs) nanostructures have recently gained extensive worldwide attentions because of their remarkable optoelectronic properties and fast developments. However, intrinsic instability against environmental factors-i.e., temperature, humidity, illumination, and oxygen-restricted their real-life applications. HPs are typically synthesized as colloids by employing organic solvents and ligands. Consequently, the precise control and tuning of complex 3D perovskite morphologies are challenging and have hardly been achieved by conventional fabrication methods. Here, we combine the benefits of self-assembly of biomolecules and an ion exchange reaction (IER) approach to customize HPs spatial shapes and composition. Initially, we apply a biomineralization approach, using biological templates (such as biopolymers, proteins, or protein assemblies), modulating the morphology of MCO3 (M = Ca2+, Ba2+) nano/microstructures. We then show that the morphology of the materials can be maintained throughout an IER process to form surface HPs with a wide variety of morphologies. The fabricated core-shell structures of metal carbonates and HPs introduce nano/microcomposites that can be sculpted into a wide diversity of 3D architectures suitable for various potential applications such as sensors, detectors, catalysis, etc. As a prototype, we fabricate disposable humidity sensors with an 11-95% detection range by casting the formed bio-templated nano/micro-composites on paper substrate.

Tunable nanofluidic device for digital nucleic acid analysis.

Nano/microfluidic-based nucleic acid tests have been proposed as a rapid and reliable diagnostic technology. Two key steps for many of these tests are target nucleic acid (NA) immobilization followed by an enzymatic reaction on the captured NAs to detect the presence of a disease-associated sequence. NA capture within a geometrically confined volume is an attractive alternative to NA surface immobilization that eliminates the need for sample pre-treatment (e.g. label-based methods such as lateral flow assays) or use of external actuators (e.g. dielectrophoresis) that are required for most nano/microfluidic-based NA tests. However, geometrically confined spaces hinder sample loading while making it challenging to capture, subsequently, retain and simultaneously expose target NAs to required enzymes. Here, using a nanofluidic device that features real-time confinement control via pneumatic actuation of a thin membrane lid, we demonstrate the loading of digital nanocavities by target NAs and exposure of target NAs to required enzymes/co-factors while the NAs are retained. In particular, as proof of principle, we amplified single-stranded DNAs (M13mp18 plasmid vector) in an array of nanocavities via two isothermal amplification approaches (loop-mediated isothermal amplification and rolling circle amplification).

Nanostructured-Based Optical Readouts Interfaced with Machine Learning for Identification of Extracellular Vesicles.

Extracellular vesicles (EVs) are shed from cancer cells into body fluids, enclosing molecular information about the underlying disease with the potential for being the target cancer biomarker in emerging diagnosis approaches such as liquid biopsy. Still, the study of EVs presents major challenges due to their heterogeneity, complexity, and scarcity. Recently, liquid biopsy platforms have allowed the study of tumor-derived materials, holding great promise for early-stage diagnosis and monitoring of cancer when interfaced with novel adaptations of optical readouts and advanced machine learning analysis. Here, recent advances in labeled and label-free optical techniques such as fluorescence, plasmonic, and chromogenic-based systems interfaced with nanostructured sensors like nanoparticles, nanoholes, and nanowires, and diverse machine learning analyses are reviewed. The adaptability of the different optical methods discussed is compared and insights are provided into prospective avenues for the translation of the technological approaches for cancer diagnosis. It is discussed that the inherent augmented properties of nanostructures enhance the sensitivity of the detection of EVs. It is concluded by reviewing recent integrations of nanostructured-based optical readouts with diverse machine learning models as novel analysis ventures that can potentially increase the capability of the methods to the point of translation into diagnostic applications.

Advances in the Translation of Electrochemical Hydrogel-Based Sensors.

Novel biomaterials for bio- and chemical sensing applications have gained considerable traction in the diagnostic community with rising trends of using biocompatible and lowly cytotoxic material. Hydrogel-based electrochemical sensors have become a promising candidate for their swellable, nano-/microporous, and aqueous 3D structures capable of immobilizing catalytic enzymes, electroactive species, whole cells, and complex tissue models, while maintaining tunable mechanical properties in wearable and implantable applications. With advances in highly controllable fabrication and processability of these novel biomaterials, the possibility of bio-nanocomposite hydrogel-based electrochemical sensing presents a paradigm shift in the development of biocompatible, "smart," and sensitive health monitoring point-of-care devices. Here, recent advances in electrochemical hydrogels for the detection of biomarkers in vitro, in situ, and in vivo are briefly reviewed to demonstrate their applicability in ideal conditions, in complex cellular environments, and in live animal models, respectively, to provide a comprehensive assessment of whether these biomaterials are ready for point-of-care translation and biointegration. Sensors based on conductive and nonconductive polymers are presented, with highlights of nano-/microstructured electrodes that provide enhanced sensitivity and selectivity in biocompatible matrices. An outlook on current challenges that shall be addressed for the realization of truly continuous real-time sensing platforms is also presented.

Biomolecules incorporated in halide perovskite nanocrystals: synthesis, optical properties, and applications.

Halide perovskite nanocrystals (HPNCs) have emerged at the forefront of nanomaterials research over the past two decades. The physicochemical and optoelectronic properties of these inorganic semiconductor nanoparticles can be modulated through the introduction of various ligands. The use of biomolecules as ligands has been demonstrated to improve the stability, luminescence, conductivity and biocompatibility of HPNCs. The rapid advancement of this field relies on a strong understanding of how the structure and properties of biomolecules influences their interactions with HPNCs, as well as their potential to extend applications of HPNCs towards biological applications. This review addresses the role of several classes of biomolecules (amino acids, proteins, carbohydrates, nucleotides, etc.) that have shown promise for improving the performance of HPNCs and their potential applications. Specifically, we have reviewed the recent advances on incorporating biomolecules with HP nanomaterials on the formation, physicochemical properties, and stability of HP compounds. We have also shed light on the potential for using HPs in biological and environmental applications by compiling some recent of proof-of-concept demonstrations. Overall, this review aims to guide the field towards incorporating biomolecules into the next-generation of high-performance HPNCs for biological and environmental applications.

Additive manufacturing leveraged microfluidic setup for sample to answer colorimetric detection of pathogens.

Colorimetric readout for the detection of infectious diseases is gaining traction at the point of care/need owing to its ease of analysis and interpretation, and integration potential with highly specific loop-mediated amplification (LAMP) assays. However, coupling colorimetric readout with LAMP is rife with challenges including, rapidity, inter-user variability, colorimetric signal quantification, and user involvement in sequential steps of the LAMP assay, hindering its application. To address these challenges, for the first time, we propose a remotely smartphone-operated automated setup consisting of (i) an additively manufactured microfluidic cartridge, (ii) a portable reflected-light imaging setup with controlled epi-illumination (PRICE) module, and (iii) a control and data analysis module. The microfluidic cartridge facilitates sample collection, lysis, mixing of amplification reagents stored on-chip, and subsequent isothermal heating for initiation of amplification in a novel way by employing tunable elastomeric chambers and auxiliary components (heaters and linear actuators). PRICE offers a new imaging setup that captures the colorimetric change of the amplification media over a plasmonic nanostructured substrate in a controlled and noise-free environment for rapid minute-scale nucleic acid detection. The control and data analysis module employs microprocessors to automate cartridge operation in tandem with the imaging module. The different device components were characterized individually and finally, as a proof of concept, SARS-CoV-2 wild-type RNA was detected with a turnaround time of 13 minutes, showing the device's clinical feasibility. The suggested automated device can be adopted in future iterations for other detection and molecular assays that require sequential fluid handling steps.

Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

Additively manufactured multiplexed electrochemical device (AMMED) for portable sample-to-answer detection.

Portable sample-to-answer devices with applications in point-of-care settings have emerged to obviate the necessity of centralized laboratories for biomarker analysis. In this work, a smartphone-operated and additively manufactured multiplexed electrochemical device (AMMED) is presented for the portable detection of biomarkers in blood and saliva. AMMED is comprised of a customized portable potentiostat with a multiplexing feature, a 3D-printed sample collection cartridge to handle three samples of saliva and blood at the same time, a smartphone application to remotely control the potentiostat, and a 3D-printed-based multiplexed microfluidic electrochemical biosensor (test chip). Here, by employing additive manufacturing techniques, a simple, cleanroom-free, and scalable approach was proposed for the fabrication of the test chip. Moreover, these techniques can bring about easy integration of AMMED components. Additionally, the test chip can be compatible with different affinity-based bioassays which can be implemented in a multiplexed manner for detection. The AMMED components were successfully characterized in terms of electrochemical and fluidic performance. Particularly, to demonstrate the biosensing capabilities of the device, the spike protein of the SARS-CoV-2 omicron variant and a well-established aptameric assay were selected as the representative biomarker and the bioassay, respectively. The proposed device accurately and selectively detected the target of interest in a rapid (5 min) and multiplex manner with a dynamic detection range of 1-10 000 pg ml-1 in different media, and the clinical feasibility was assessed by several saliva patient samples. AMMED offers a versatile sample-to-answer platform that can be used for the detection of various biomarkers present in biofluids.