Review www.advmat.de The Microbiome Meets Nanotechnology: Opportunities and Challenges in Developing New Diagnostic Devices Celia Fuentes-Chust, Claudio Parolo, Giulio Rosati, Lourdes Rivas, Karla Perez-Toralla, Stéphanie Simon, Itziar de Lecuona, Christophe Junot, Jonel Trebicka, and Arben Merkoçi* Since then, the paucity of knowledge on Monitoring of the human microbiome is an emerging area of diagnostics the links between human microbiota and for personalized medicine. Here, the potential of different nanomaterials human health have been increasingly and nanobiosensing technologies is reviewed for the development of novel addressed in academia, the clinic and diagnostic devices for the detection and measurement of microbiome-related industry. biomarkers. Moreover, the current and future landscape of microbiome-based diagnostics is defined by exploring the advantages and disadvantages of cur- 1.1. Commensal Microbes: A Necessary rent nanotechnology-based approaches, especially in the context of developing and Ever-Changing Organ point-of-care (PoC) devices that would meet the international guidelines known as REASSURED (Real-time connectivity; Ease of specimen collection; A recent study has estimated that in and on the human body, there is a 1:1 ratio of Affordability; Sensitivity; Specificity; User-friendliness; Rapid & robust opera- microbial and human cells.[2] These micro- tion; Equipment-free; and Deliverability). Finally, the strategies of the latest bial cells form the human microbiome. international scientific consortia working in this field are analyzed, the current Although most (60%) microbial cells in microbiome diagnostics market are reported and the principal ethical, legal, humans are located in the gut, others can and societal issues related to microbiome R&D and innovation are discussed. be found in areas such as the gastrointes- tinal tract, the skin, the genital surfaces and the oral cavity (Figure 1).[3] The inter- actions between humans and the human microbiome, known 1. Introduction as mutualistic symbiosis, has been shaped through millions of Since the dawn of evolution, microorganisms have co-evolved years of co-evolution that has strengthened both parties.[4] On with every plant and animal species, determining the physi- one hand, humans provide their commensal microbes with ology and health condition of their hosts, including modern nutrients and shelter; on the other, these microbes assist their humans and our ancestors. As affirmed by Lederberg & human hosts by performing location-specific functions such as McCray nearly 2 decades ago: “the microbiome is an eco- immune defense (Figure 1). In fact, the microbiota has together logical community of commensal, symbiotic, and pathogenic been considered as a separate “organ”.[5] However, from birth, microorganisms that literally share our body space and have said “organ” is constantly evolving, as it is influenced by been all but ignored as determinants of health and disease”.[1] genetic as well as by environmental factors (e.g., diet, stress, or C. Fuentes-Chust, Dr. C. Parolo, G. Rosati, Dr. L. Rivas, Prof. A. Merkoçi Dr. I. de Lecuona Nanobioelectronics and Biosensors Group Bioethics and Law Observatory -UNESCO Chair in Bioethics-Department Institut Català de Nanociència i Nanotecnologia (ICN2) of Medicine UAB Campus, Bellaterra, Barcelona 08193, Spain University of Barcelona E-mail: arben.merkoci@icn2.cat Barcelona 08007, Spain Dr. K. Perez-Toralla, Dr. S. Simon, Dr. C. Junot Prof. J. Trebicka Université Paris-Saclay, CEA, INRAE Department of Internal Medicine I Département Médicaments et Technologies pour la Santé (MTS) Goethe University Frankfurt SPI Theodor-Stern-Kai 7, 60590 Frankfurt, Germany Gif-sur-Yvette cedex 91191, France Prof. J. Trebicka The ORCID identification number(s) for the author(s) of this article European Foundation for the Study of Chronic Liver Failure can be found under https://doi.org/10.1002/adma.202006104. Travesera de Gracia 11, Barcelona 08021, Spain Prof. A. Merkoçi © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH. ICREA This is an open access article under the terms of the Creative Commons Institució Catalana de Recerca i Estudis Avançats Attribution-NonCommercial-NoDerivs License, which permits use and Pg. Lluís Companys 23, Barcelona 08010, Spain distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. The copyright line for this article was changed on 21 October 2021 after original online publication. DOI: 10.1002/adma.202006104 Adv. Mater. 2021, 33, 2006104 2006104 (1 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Figure 1. Simplified overview of commensal microbes by body location, showing their corresponding functions and links to human health. Imbalances in local microbial populations have been linked to myriad diseases. Some examples of involved bacteria in these conditions are shown. drug use).[6,7] Such factors modulate the number and relative gut-flora composition by non-conventional gut bacteria pro- abundance of microbiome species, known as the alpha diver- vokes secretion of the antibacterial peptides alpha defensins sity. Based on this premise, El Rakaiby et al. referred to the by host innate immune cells, and another found that con- human microbiome as “a cloud of genetic information acces- ventional gut flora induce Paneth cells of the small intes- sory to the stable human genome”.[8] tine to express the antibacterial protein angiogenin-4, which targets gram-positive bacteria.[9,10] Despite a certain degree of transitory variability, which itself varies among humans, 1.2. Clinical Impact researchers have demonstrated that for roughly 60% of the species in an individual’s microbiome, the population The continuous communication among humans, their remains constant over a 5-year period.[11–13] Accordingly, microbiome and their environment largely dictates short- the corresponding population data can be used as reference term variability in alpha diversity, which precludes defini- values, such that any significant changes in the relative or tion of a stable or default microbiome corresponding to good absolute population of one or more species could indicate a health.[8] For instance, one study found that alteration of the pathologic event. Adv. Mater. 2021, 33, 2006104 2006104 (2 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Researchers have already associated alterations in the micro- metagenomic studies to find a relation to microbiota composi- biome to various diseases (e.g., colorectal cancer, celiac disease, tion, whereby dead or transient species can be ruled out. Thus, cirrhosis and inflammatory bowel disease), conditions (e.g., obe- the combination of metagenomics with metatranscriptomics, sity and allergies) and clinical parameters (e.g., longevity).[14–18] metaproteomics and metabolomics generates a multi-omics For instance, strengthening of the natural commensal micro- approach that can give a much better understanding of specific biota has been found to play a significant role in the immune microbiome behavior.[27] response towards influenza virus.[19] Eventually, monitoriza- As emphasized by Petrosino et al., numerous studies have tion and use of specific probiotics might be very useful for the now revealed microbiome–disease associations, making the assessment of the diagnosis and prognosis of virus infections, microbiome a key component of precision medicine.[33] This is as in the current SARS-CoV-2 outbreak.[20–24] Another example for example the case in the fields of inflammatory bowel dis- involves the microbiome gut-brain axis (Figure 1), whose con- eases; liver diseases such as cirrhosis, rheumatic diseases such stant communication pathway between nervous and endocrine as arthritis, oncology and response to anticancer chemothera- systems modulates our behavior and our mental state (e.g., pies.[25,34–38] In another example, Pedersen et al. have shown causing or preventing anxiety and depression).[16] At last, by re- that the serum metabolome of insulin-resistant individuals is examining whole-genome and whole-transcriptome sequencing characterized by increased concentrations of branched-chain studies in The Cancer Genome Atlas (TCGA) of 33 types of amino acids, which correlate to a gut microbiome having an cancer from treatment-naive patients, Poore et al. have found enriched biosynthetic potential for these acids.[39] Integrating unique microbial signatures in tissue and blood within and all this information is important to understand how the between most major types of cancer, suggesting that these microbiome interacts with the host in response to changes in microbial nucleic acids could have a diagnostic value.[25] Thus, the microbial population, in diet, in aging or in health or dis- due to all the foreseeable implications the microbiome has on ease. Thus, the diagnostic question has evolved from simply human health, our group and many others consider that moni- determining the presence or absence of concrete species in toring of the microbiome, beyond its value for fundamental sci- the microbiome, to characterizing how certain species relate to entific research, represents a novel diagnostic strategy for per- each other, to the host and to health or disease. sonalized medicine. Although there are a large number of studies mainly based on metagenomic analyses, a review of the literature reveals a growing number of works that are produced by integrating 1.3. Microbiome R&D: From Technology to Signatures multi-omic analyses. These studies can make it possible to of Diagnostic Significance document the links between microbiome and pathologies. For example, Franzosa et al. performed untargeted metabo- Over the past few decades, the tools available to study the micro- lomic and shotgun metagenomic profiling of cross-sectional biome have become increasingly sophisticated and diverse, stool samples from discovery and validation cohorts of Crohn’s from the first culture-based and DNA-sequencing techniques disease, ulcerative colitis and control patients.[34] By inte- (the 1970s), to next-generation sequencing (the early 2000s), to grating metagenomics and metabolomics data, they identified the latest omics-based approaches.[26] For example, previously, 122 robust associations between differentially abundant species using culture-based techniques, researchers could isolate only and well-characterized differentially abundant metabolites, thus 30% of the species of the gut microbiota; in contrast, omics- suggesting potential diagnostic and therapeutic targets.[34] In based approaches have revealed a nearly complete view of the the same line, Price et al. followed 132 subjects (with Crohn’s alpha diversity.[27–30] The current gold-standard technique for disease, with ulcerative colitis, or without inflammatory bowel studying the human microbiome is metagenomics, which ena- disease, i.e., controls) for one year each to generate integrated bles discrimination of bacterial and archaeal DNA from host longitudinal molecular profiles of host and microbial activity DNA, based on measurement of prokaryote-specific genes during disease (up to 24 time points each; in total 2965 stool, known as 16S ribosomal RNA genes. Metagenomics results biopsy, and blood specimens).[35] They observed a character- enable taxonomic classification of microbiota at a given point istic increase in facultative anaerobes at the expense of obli- in time, which can then be used to generate a profile of alpha gate anaerobes, as well as molecular disruptions in microbial diversity to facilitate comparison of different microbial sam- transcription (for example, among Clostridia), metabolite pools ples: for instance, from the same individual at different times, (acylcarnitines, bile acids, and short-chain fatty acids), and or from distinct individuals or cohorts.[31] levels of antibodies in host serum.[35] Of course, the translation Correlating alpha diversity with a given condition or disease of these findings in diagnostic tools will require comparative is not trivial, chiefly because not all the bacteria present at a analyses that will be enabled by data access through data reposi- given time are implicated in the condition or disease in ques- tories, and also further validation studies. tion.[32] For instance, a detected species might be only tran- Multi-omic analyses have also the potential to shed light on siently present when the sample is collected, or the cells may the mechanisms of interaction between the microbiota and the even be dead but are still detected. This limitation has been par- host. This has for example been achieved by Rosser et al. who tially offset in the past 5 years, through the use of techniques highlighted the role of microbiota-derived metabolites to sup- such as metatranscriptomics (RNA), metaproteomics (proteins), press arthritis by working on samples obtained from patients and metabolomics (metabolites), which provide information on and an animal model.[37] They showed that stool butyrate different levels of distinct molecules within a population of dif- levels are reduced in patients with rheumatoid arthritis com- ferent bacterial species.[27] The results are then compared with pared to healthy controls, and that supplementation with Adv. Mater. 2021, 33, 2006104 2006104 (3 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Table 1. Microbiome related studies on therapeutics, diagnostics, and microbiome. The Evivo test relies on a change in fecal pH when involving (bio)sensors as an indicative of the growing trend of these bio- the gut is first colonized by this bacterium; thus, results are sensors in clinical trials. quickly obtained for babies suffering from low levels of Bifido- bacterium. Another interesting product with potential for micro- Number of microbiome Total Completed Ongoing Terminated/ biome diagnostics is MinIon (Oxford Nanopore Technologies), a studies in clinicaltrials.gov number (recruiting) withdrawn/ (25. October 2020) suspended portable, real-time DNA/RNA sequencer. Although this device was not designed for microbiome analysis per se, it can process Therapeutics 1023 285 499 51 microbiome samples and classify the microbiota by phyla and Diagnostics 370 72 198 16 genera. It reads electrical signals that are generated by the dif- Involving Sensors/biosensors 16 4 12 0 ferent nucleotides when passing through a nanopore. It enables in situ sequencing, de novo sequencing, targeted sequencing, butyrate suppresses arthritis severity in a mouse model. They metagenomics studies, and epigenetics studies, amongst others. also demon­strated that butyrate increases the concentration of Nevertheless, MinIon is only intended for trained users. 5-hydroxyindole acetic acid, a metabolite derived from serotonin that activate aryl-hydrocarbon receptor in regulatory B cells.[37] Microbiome derived signature can also have a diagnostic 1.3.2. International Consortia value, as emphasized by the study performed by Oh et al., who identified diagnostic signatures for fibrosis from stool metagen- Many international projects have been launched to charac- omic and metabolomic profiling that, when combined with terize the human microbiome and analyze its role in human serum aspartate transaminase levels, distinguishes cirrhosis in health and disease. In 2007, researchers in the Human Micro- mixed fibrosis cohort.[36] The cohort included 163 participants biome Project, funded by the National Institutes of Health encompassing non-NAFLD (non-alcoholic fatty liver disease) (NIH), used metagenomics to study commensal microbiota in controls, NAFLD-cirrhosis patients, and their first-degree rela- healthy individuals at five anatomic locations: the nasal cavity, tives. The gut metagenomic and metabolomics signatures com- the oral cavity, the skin, the gastrointestinal tract and the uro- prised 19 discriminatory species (among the 310 microbial spe- genital tract.[139] After they were unable to define a standard cies identified) and 17 metabolites (among the 435 quantified healthy microbiome, NIH researchers are now exploring host- ones) that accurately detected NAFLD-cirrhosis in the proband microbiome interplay in three clinical scenarios: pregnancy, cohort, respectively. inflammatory bowel disease and diabetes.[140] In the MetaHIT The growing interest in the human microbiome as a deter- project, which ended in 2012, researchers studied the intestinal minant in health and disease, the creation of new scientific tract microbiota of 1000 healthy people, ultimately dividing consortia, and increasing participation in studies by members the cohort into three classes, or enteromes, defined by the of the public (see Section 1.3.1 and 1.3.2), together set the stage dominant bacterium in their respective microbiota.[141] Other for the human microbiome to be included in precision and per- platforms, including the Human Food Project and the Earth sonalized medicine. Microbiome Project, joined together to create the American Gut Project (AGP), an open research platform that accepts micro- biota samples from anyone in the world.[142] They aim to charac- 1.3.1. The Microbiome Diagnostics Market and Current Products terize the taxonomic and functional diversity of the microbiome and its diversity across human populations to define a baseline The microbiome therapeutics & diagnostics market is healthy microbiome. expected to grow from $506 million in 2022 to $899 million Several international projects already employ multi-omics by 2025 (a compound annual growth rate of representing an approaches.[142] For example, by using metagenomics and metabo- 22.1%).[138] This prediction suggests growing acceptance of lomics, researchers in the open-source project AGP seek to corre- microbiome-based products by the general public. This market late participants’ samples with clinical parameters (e.g., age) and can be divided into two segments: therapeutics and diagnos- lifestyle parameters (e.g., dietary, smoking, or drinking habits). tics. Although both are slated for expansion, diagnostics are Our groups are currently developing a nanobiosensor for expected to grow the most, due mainly to the discovery of detection and monitoring of biomarkers for cirrhosis and microbiome-related biomarkers for oncology. acute-on-chronic liver failure (ACLF), as part of the European Presently, there are commercially available microbiome- consortium Microb-Predict, which is integrating multi-omics related tests and analyses; however, these products are not recom- data from over 10 000 subjects from different cohorts to develop mended for use alone as diagnostics. Today’s analyses, such as novel diagnostics and treatment strategies for these conditions. uBiome, analyze the alpha diversity of the microbiome at one or Specifically, we aim to create a diagnostic platform to detect more timepoints, to enable temporal monitoring of microbiome ACLF at an earlier stage than presently possible, by monitoring composition and correlation of it to life events. These results the patient’s microbiome. can be also analyzed by a healthcare provider to help to deter- mine a diagnosis. A simpler technology is provided by Evivo, which produces a PoC test to screen newborn babies for the 1.4. Clinical Trials Landscape in the Interface of Microbiome commensal bacterium Bifidobacteria infantis. Newborns acquire and Nanotechnology their gut microbiome during passage through the birth canal, which that inoculates the baby with Bifidobacterium, protecting it The gut microbiome may serve as biomarker for disease pro- from harmful bacteria and serving as the basis for the remaining gression, severity and treatment response.[40] Indeed, diagnostic Adv. Mater. 2021, 33, 2006104 2006104 (4 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de biomarkers deriving from the gut microbiome have been tested maximum information possible. In order to include the human in different diseases, such as diabetes, colorectal cancer, and microbiome, some aspects need to be considered. cirrhosis.[36,41–45] Microbiome may also determine the treatment response and thereby guide patients management, as shown a. As previously mentioned, obtaining a list of species and for epithelial cancer and melanoma, where the use of immune metabolites contained in a particular microbiome by means therapy is a move towards targeting the microenvironment by of metagenomics or other omics is not entirely useful. employing nanotechnology.[46–50] Therefore, intervention on There still exists the need of analyzing the results and the microbiome are being extensively explored as indicated by relating them to the patient’s lifestyle, which is an exhaus- more than 1000 registered trials, with half of them ongoing tive work. Besides, it is unlikely that healthcare centers (Table 1). could implement routine metagenomic analyses due to Yet, analyzing the gut microbiome is not always easy, since the high cost of the equipment and the highly trained per- also the location of the microbiome is important as shown sonnel required. in gastroenterology and hepatology.[51,52] Patients with dif- b. Nowadays routine analyses regarding microbiological sam- ferent diseases exhibit both disease-specific changes, as well ples rely on culture-based approaches, which are slow as non-specific shared responses in their gut microbiome.[53] and only provide information about few species, and not Therefore, one should first identify robust disease-specific sig- metabolites. natures, and even more relevant the mapping of the micro- biome changes during the specific disease, which should be Instead, future precision and personalized medicine should independent of other confounding factors. This is still difficult establish a panel of biomarkers for each microbiome-related to foresee, since the confounding effect size of different known condition in order to simplify and fasten routine analyses. technical and biological factors ranges between 10%–15%.[40] These biomarkers would include cells, nucleic acids and other Moreover, large-scale longitudinal data from across the world molecules, and the technology used for their detection should are required to account for the confounding factors, since the adapt to a convenient format in order to bring these analyses to variation in the genus composition of gut microbiome by the the everyday life with a low cost. specific diagnosis may change only by including or removing We think that point-of-care (PoC) nanobiosensors would be confounders, such as alcohol consumption or diet. Another suitable for this purpose. Importantly, the World Health Organ- major confounder for the gut microbiome composition are ization has established criteria to facilitate access to, and use of, drugs, and not only antibiotic, but also non-antibiotic drugs, new PoC devices by all types of patients in developing and devel- one in four of which may change at least one gut species and oped countries, known collectively as REASSURED (Real-time this is likely underestimated.[54] Some of the non-antibiotic connectivity, Ease of specimen collection, Affordability, Sensi- drugs may induce even bacterial resistance, while others may tivity, Specificity, User-friendliness, Rapid and Robust opera- improve diversity.[54,55] tion, Equipment-free and Deliverability).[57] Thus, any new PoC Having said this, it is not surprising that a large number of device for microbiome-based diagnostics should ideally adhere studies have used and are currently using the microbiome for to the REASSURED criteria. Within the realm of PoC sensors, diagnostic purposes (Table 1). Through the revolution in the nanobiosensors harness the properties of nanomaterials (e.g., microbiome field, different options are now available to iden- higher surface area, their optical and electrical properties and tify diseases in microbiome profiles, as shown recently by the their high tunability) to achieve superior analytical and usability Microbiome Search Engine.[56] Moreover, integration with other performances.[58–60] Considering the unique characteristics of omics data may facilitate that the dream of “personalized” med- nanomaterials is of great importance when orchestrating the icine becomes reality.[33] detection of the very diverse biomarkers involved in the micro- Still robust gut microbiome biomarkers are feasible, as biome, such as cells, lipids, proteins, nucleic acids and small demonstrated recently for colorectal cancer across large geo- molecules. graphic distances and different studies, but the applicability The huge amount of data generated by multi-omics requires the expertise of nanotechnology.[44] In fact, there are approaches has enabled discovery of microbiome biomarkers very few studies using sensors and biosensors to analyze the associated with specific conditions and diseases, which in turn microbiome (see Table 1). Especially in these studies, none of are providing a window to new novel diagnostics strategies.[70] them is testing or validating a sensor for the detection of the Thus, one could envision development of sensitive and fast microbiome, but those detecting glucose, volatiles, sleep, move- sensors for hospital- or home-based monitoring of microbiome- ment, heart rate variability and endotoxin levels. The field of related biomarkers, which could ultimately change diagnosis microbiome currently urges for easy-to-use tools in the deter- and daily life for patients suffering from various diseases, mination of the diagnostic and prognostic signatures, and similarly to how glucometers have changed the lives of diabetic nanotechnology may develop the answer. patients (see Scheme 1). We believe that nanobiosensor-based PoC devices for detec- tion of biomarkers could replace the current gold-standard 2. The Need for New PoC Devices and New techniques of metagenomics and culture-based analyses, which are expensive and time-consuming. Thus, we review here nano- Sensors biosensors that respond to the more general definition of PoC The future of precision and personalized medicine would devices, meaning that they can provide results wherever the require the access to in-depth routine analyses that provide the patient needs them (e.g., at home or at the hospital). However, Adv. Mater. 2021, 33, 2006104 2006104 (5 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Scheme 1. Nanobiosensing for detection of validated microbiome-related biomarkers based on multi-omics data. Point-of-care devices based on nanobiosensors could enable prevention, early diagnosis and monitoring of microbiome-related conditions according to diverse clinical parameters (e.g., symptoms, antibiotics use, age, family medical history) and lifestyle parameters (e.g., diet, stress, or drug use). due to the variety of microbiome-related biomarkers, ranging 3. PoC Nanobiosensors and Microbiome-Related from small molecules to whole bacteria (see Table 2), and to Biomarkers their complicated analysis, which involves special equipment or specific sample treatment methods, meeting REASSURED As the microbiome is constantly changing, PoC sensors for criteria is not always feasible.[57] Therefore, we also discuss microbiome-biomarkers should comply with extra moni- nanotechnologies that we refer to here as “quasi- PoC”, that toring and multiplexing capabilities. In fact, obtaining might require to be used in a laboratory or to be interpreted clinically-relevant information on a patient’s status may by trained personnel. Although these devices or technologies require simultaneous, real-time monitoring of multiple bio- cannot be used at home or other locations directly by patients, markers such as metabolites, sugars, lipids, nucleic acids, they could be implemented in healthcare centers for efficient, peptides and proteins.[71] Additionally, PoC sensors must cost-effective, routine testing. overcome the challenges faced by other biosensors: namely, Table 2. Examples of microbiome-related biomarkers and availability of dedicated (nano)biosensors for the diagnosis of different diseases. The type of molecule and weight/size column serves to illustrate on the very different kinds of potential biomarkers. mRNA: messenger RNA. PoC Technology Disease Biomarker Type of molecule Weight/Size Sample Ref Already exists Lateral flow Intestinal inflammation Calprotectin Zinc-binding protein 35.6 kDa Stool [61] Already exists Lateral flow SARS-CoV-2 Human IgG/IgM Immunoglobulin 150 kDa Saliva [23] Optimizable Lateral flow Tuberculosis Mycobacterial lipoarabinomannan Lipoglycan 17.4 kDa Urine [62,63] (LAM) Optimizable Paper-based sensor Clostridium difficile infection (CDI) Calprotectin mRNA Nucleic acid – Stool [64] CXCL5 mRNA – IL-8 mRNA – Not available – Equine grass sickness (EGS) 4-cresyl-sulfate Organic compound 188.2 kDa Urine [65] Hippurate 179.2 kDa TMAO 75.1 kDa O-acetyl carnitine 203.2 kDa Not available – Colorectal cancer (CRC) Matrix-metalloproteinase-9 (MMP-9) Enzyme 92 kDa Stool [66] Not available – Multiple sclerosis Lipid 654 Lipopeptide 653.5 kDa Stool [67] Not available – Lung cancer Capnocytophaga Nucleic acid 0.3–0.5 µm Saliva [68] Veillonella Whole cell 0.3–0.5 µm Not available – Cirrhosis Kynurenic acid/Quinolinic acid Metabolites <500 Da Plasma [69] Not available Cirrhosis Molecular signature including 17 Metabolites <500 Da Stool [36] discriminatory metabolites Molecular signature related to 19 Nucleic acid sequences discriminatory bacterial species Adv. Mater. 2021, 33, 2006104 2006104 (6 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de that small-molecule biomarkers present steric hindrance the type of quantum dot employed, making them ideal labels caused by the congestion of the molecules by its surrounding for the multiplexed detection of biomarkers including different ligands, and that complex media can pose challenges in terms mycotoxins, food contaminants and viruses.[58,80,81] Finally, of specificity and ease-of-use. In this context, nanomaterials more recently, research groups employed upconverting nano- offer a great opportunity to provide novel PoC sensors with particle (UPTs) to achieve even greater sensitivity, although we the required performance. Here, we describe the advantages must point out that their high cost may prevent their use at a of nanomaterials as the basis of PoC sensors for detection large scale.[82] of microbiome-related biomarkers. These include relatively Beyond optical detection, nanomaterials serve other roles in large surface areas, easy functionalization, and unique optical LFAs. For example, they have been used as signal generators to and electrical properties. improve sensitivity in diverse readout strategies (using chromo- phores and fluorescent dyes) such as surface-enhanced Raman spectroscopy (SERS) over the usual or standard LFA.[79,83–85] 3.1. Lateral-Flow Assays Intriguingly, other different nanomaterials can be combined to enhance sensitivity, as has been shown through use of gra- 3.1.1. Overview of Lateral-Flow Assays phene to quench quantum dots as a revealing agent in LFAs for detection of bacteria, or the use of magnetic nanoparticles for Most PoC nanobiosensors are based on lateral-flow assays preconcentration and purification of the sample.[86–90] (LFAs), the best-known of which is the home pregnancy test, which detects or measures the hormone human chorionic gon- adotropin (hCG) in urine. Given their ease-of-use, LFAs have 3.1.3. LFA Challenges been extended for detection or measurement of various ana- lytes such as toxins, pathogens, pesticides, metal ions, drugs These and many other examples suggest the utility of LFAs and proteins.[58–60] Importantly, LFAs meet nearly all the REAS- for simple, microbiome-related applications. However, there SURED criteria;[57] however, their sensitivity remains limited. A remain major challenges to this end. Firstly, multiple detection typical LFA comprises a paper strip onto which a liquid sample of different types of molecules is complicated due to the use of is added, which generates a chemical reaction whose result is bioreceptors of different natures under a single measurement discernable by the naked eye (e.g., by color change). Specifi- and conditions. Secondly, microbiome analyses often require cally, the sample moves via capillary action through different simultaneous detection of more than ten analytes (for which zones of the strip, each of which is dedicated to a specific func- we saw the possible use of quantum dots as multi-color labels), tion (e.g., sample loading and target labelling; migration and which is not trivial in terms of sensor fabrication and sensor analyte detection; and sample pumping). These assays employ readout, even when using a smartphone. Thirdly, analyses of signal amplifiers conjugated to bioreceptors (e.g., antibodies, microbial nucleic acids often entail detection at extremely low nucleic acids or enzymes), which enable recognition of the concentrations (copy numbers), which would demand an inte- target analyte(s) (Figure 2A).[60] grated amplification step or the use of ultra-sensitive labels (e.g., UPTs). Similarly, microbiome analyses may require detec- tion of protein analytes in very low ranges, like the femtomolar 3.1.2. Nanomaterials and LFAs range, which is still difficult to achieve. Many LFAs exploit the unique optical properties of nanoma- terials. Specifically gold nanoparticles (AuNPs) are the most 3.2. Electrochemical Sensors used label in LFAs, thanks to their easy functionalization and plasmon properties that confer them a strong red color that 3.2.1. Overview of Electrochemical Sensors is ideal for naked eye detection.[58] As alternative to AuNPs, iridium oxide nanoparticles, dyed latex beads, and carbon Another technology used for PoC diagnostic devices is elec- nanoparticles and nanotubes have also been employed as col- trochemical sensing, as in the widely successful example of orimetric labels in LFA.[60,76] In order to improve the LFA sen- the glucometer and much beyond. Although electrochemical sitivity yet using standard AuNPs, some developers have also PoC sensors require a reader, making them less portable than taken advantage of their catalytic properties, used them as LFA-based PoC sensors, their superior analytical performance carrier for enzymes, and used their quenching properties in compared to the latter (greater sensitivity, quantitative meas- fluorescence-based measurements.[77–79] urements and real-time monitoring) is ideal for specific appli- Instead, applications requiring a higher degree of sensitivity cations, such as monitoring of glucose levels.[91,92] The most often rely on fluorescence signals. In this arena the most used common electrochemical techniques used in PoC sensors are nanoparticles are semiconducting or carbon-based quantum cyclic voltammetry,[93–95] differential pulse voltammetry,[91] dots, silica beads and liposomes (Figure 2B).[58,80,81] Comparing chronoamperometry,[96,97] electrochemical impedance spectros- them to conventional organic dyes, their higher quantum yield copy[98,99] and field-effect transistor (FET)-based sensing.[100,101] and stability make them ideal labels for PoC applications, The first three techniques are used to analyze the currents although they do require a fluorescence reader. Another useful obtained in response to fluctuations in the concentration of a quality of quantum dots is the possibility to excite them with a redox reporter, which can be quantitatively related to the con- UV light and record different emission spectra depending on centration of the target analyte.[102] In contrast, impedance Adv. Mater. 2021, 33, 2006104 2006104 (7 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Figure 2. Point-of-care biosensing devices amenable to microbiome analyses. A) Multiplexing: Schematic of a multiplex lateral-flow immunoassay (LFA) for multiple mycotoxins. Adapted with permission.[72] Copyright 2014, American Chemical Society. B) Real-time connectivity: Smartphone-based fluorescent LFIA platform for highly sensitive PoC detection of Zika virus non-structural protein 1. Reproduced with permission.[73] Copyright 2019, Elsevier. C) Monitoring (C&D): In-vivo continuous glucose monitoring in healthy human subjects. Left: screen-printed array fixed onto a volunteer’s forearm and connected to a potentiostat. Right: values of interstitial fluid-borne glucose as read from extracted blood (red) or through the skin (black). Adapted with permission.[74] Copyright 2018, The Authors, published by Springer Nature. D) Epidermal microfluidic sweat-monitoring device before (left) and after (right) injection of artificial sweat. Lactate, glucose, creatinine, pH, and chloride ions in the sweat sample are monitored and integrated near-field communication (NFC) electronics enabled smartphone-based analysis. Reproduced with permission.[75] Copyright 2016, AAAS. spectroscopy and FET-based sensing detect the analyte performance.[108,109] Since its discovery in 2004,[110] graphene has according to changes in the electronic properties of the sensor attracted considerable attention owing to its remarkable proper- surface induced upon specific binding of the analyte to it. ties, including high carrier mobility and capacity, an ultrathin form factor, an ambipolar field effect, and highly tunable con- ductance.[111,112] Graphene has been extensively explored in 3.2.2. Nanomaterials and Electrochemistry the development of high sensitivity biosensors based on FETs (gFETs), by using the resistivity variation in the gFETs due to As with LFA-based sensors, analytical performance in electro- the binding of the target molecule to the bioreceptor on its chemical sensors has been optimized through strategic use of surface.[101,113] nanomaterials, either as transducers in the sensing platform Transition metal dichalcogenides, 2D layered materials or as electrochemical reporters. Examples of such transducers with ultrathin structures such as molybdenium disulfide include electrodes fabricated from (or modified with) AuNPs, is attracting a lot of interest too. This semiconductor has a silver nanoparticles (AgNPs), carbon nanotubes, graphene or tunable bandgap and piezoelectric properties, which allow magnetic nanoparticles (Figure 2C, Figure 3A,B).[91,103] Spe- to improve the sensitivity of these sensors and make them cifically, AuNPs and AgNPs have been widely used as trans- more stable.[114] The intercalation of specific elements in ducer components in electrochemical assays due to their the structure of these materials allows precision function- high conductivity and easy functionalization. Furthermore alization, lowering its performance dependence by environ- their high stability and homogenous distribution (achieved mental variables. Moreover, the ultrathin structure of these using simple synthesis protocols such as the Turkevich materials gives them excellent mechanical properties such as method)[104] allow for the fabrication of electrochemical sen- flexibility and strain resistance, both very sought character- sors using easy and low-cost methods such as screen or inkjet istics for wearable biosensors for instance.[115] However, the printing.[91,105–107] limits of these materials are usually bound to their synthesis More recently, researchers have been working on exploiting and to the production of a truly uniform nanosheet able to the high electron mobility and capacity of 2D nanomate- completely cover the sensor’s electrodes. Consequently, these rials such as reduced graphene oxide and molybdenum types of sensors keep costs relatively high with respect to the disulfide to enhance electrodes’ conductivity and analytical standard ones. Adv. Mater. 2021, 33, 2006104 2006104 (8 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Figure 3. Examples of nanobiosensing technologies with potential for microbiome-related diagnostics. A) Wireless, biotransferrable, graphene-based nanosensor (left). Graphene is printed onto bioresorbable silk and contacts are formed containing an inductive resonant coil for wireless transmission (middle). Magnified schematic of the sensing element, illustrating wireless readout of the binding of pathogenic bacteria by peptides self-assembled on the graphene nanotransducer (right). Adapted with permission.[118] Copyright 2012, Springer Nature. B) CRISPR-enhanced, graphene-based, field- effect transistor (CRISPR–Chip), which enables facile, rapid and selective detection of a target sequence contained within whole intact genomic DNA. Adapted with permission.[101] Copyright 2019, The Authors, published by Springer Nature. C) Illustration of interactions between bacterial target and probe molecules on cantilever beam. Specific biomolecular interactions between target and probe molecules alter the intermolecular nanomechanical interactions within a self-assembled monolayer on one side of a cantilever beam. Adapted with permission.[119] Copyright 2013, Springer Nature. D) A conventional, colorimetric, enzyme-linked immunosorbent assay for visual detection of the target antigen (top, right). A non-conventional colorimetric ELISA or Plasmonic ELISA, whose ultra sensitivity allows the detection of the target at ultra-low concentrations with the naked eye (down, right). Adapted with permission.[120] Copyright 2012, Springer Nature. E) Highly sensitive, magnetic barcode assay for detection of tuberculosis, equipped with polymerase chain reaction (PCR) chambers, mixing channels, and a microcoil for NMR measurements. The PCR-amplified target genes from Myco- bacterium tuberculosis are captured and magnetically labeled by a pair of complementary oligonucleotides conjugated to microspheres and magnetic nanoprobes. Adapted with permission.[121] Copyright 2013, Springer Nature. Looking at the use of nanomaterials as electrochemical strategies, and reagents mixing can be implemented also reporters there are three main characteristics that must be con- within electrochemical sensors thanks to the use of similar sidered: size, functionalization and signal generation. Overall substartes as for the electrochemical paper-based analytical a successful electrochemical label should have a size in the devices (ePADs) and of nanomaterials. What is still a chal- same scale as the one of the bioreceptor (mostly in the low nm lenge is the immediacy of a LFA result if compared to the range), a surface chemistry that allows for an oriented function- need of a readout instrument and the interferences of electro- alization and the ability to generate a strong electrochemical chemically active compounds in the sample which may alter signal. All these aspects are generally found in nanoparticles, the result of the tests if not filtered or without signal normali- rather than 2D materials. Overall, AuNPs and AgNPs are the zation. With respect to the readout instrument, smartphones most used electrochemical labels in voltammetric and amper- are becoming an interesting platform for the purpose, con- ometric sensors. Specifically, the former measure the voltam- sidering how widespread they are. Conversely, with reference metric peak of Au and Ag, while the latter rely on their catalytic to interference, big testing campaign and common interfer- activities, such as the hydrogen evolution reaction of AuNPs. ence compensation in the sample of interest remain the only As for LFA, also electrochemical sensors can employ QDs in weapons against it. multiplexed sensors, thanks to the different voltammetric peaks Interestingly, in the context of microbiome-related dis- related to the different QD composition.[116] eases (e.g., Crohn’s disease), wearable electrochemical devices can be used to monitor inflammation and other physiological symptoms (Figs. 2C & 3A).[117] Considering the 3.2.3. Electrochemical Sensors Challenges nearly infinite number of possible combinations of function- alized nanomaterials, electrochemical PoC devices could, in Several of the commonly recognized advantages of LFA sen- theory, be used to detect a broad array of microbiome-related sors, such as sample filtration and purification, amplification biomarkers. Adv. Mater. 2021, 33, 2006104 2006104 (9 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de 4. Quasi-PoC Nanobiosensors with Potential expertise, making it highly amenable to PoC applications. How- in Microbiome-Related Diagnostics ever, despite this example and other widespread efforts to bring SPR sensors into PoC scenarios, these devices tend to be expen- Recent advances in nanotechnology have enabled conception sive and require specialized equipment.[129,130] If researchers of exciting new diagnostic sensors that, despite not meeting can reduce the costs and resolve the matrix-effect issues caused REASSURED criteria,[57] should provide a basis on which to by interfering components of human samples when using SPR construct future PoC devices or at least, devices for hospital biosensors, then these devices could be implemented in labora- or laboratory use. Below, we highlight representative devices tories and healthcare centers for faster and more detailed rou- with strong potential for detection of microbiome-related tine analyses. biomarkers. ELISA tests are nowadays a common tool in hospitals and healthcare centers as in vitro screening diagnostic tests. Plas- monic ELISAs exploit the innate plasmonic properties of gold 4.1. Cantilevers nanoparticles, properties which can be tuned during nanopar- ticle synthesis. For instance, De la Rica et al. managed to use Microcantilevers (or microelectromechanical systems) comprise the output of ELISA to control both the synthesis of AuNPs and a cantilever structure that measures the vibration or deflec- the growth of gold nanostars.[130,131] Thanks to the strong and tion generated by the adsorption of a molecule on its surface. sensitive optical signal generated by the plasmon of those par- Known for their high sensitivity, these devices can detect ana- ticles, they obtained outstanding detection limits (in the range lytes in the picomolar to femtomolar range (Figure 3C).[122,123] of 10−21 M) with the naked eye (Figure 3D).[130] Thus, plasmonic Nanocantilevers (cantilevers of thickness from 10 nm to 90 nm) ELISAs could be used to detect microbiome-related proteins at exhibit even better limits of detection and require less sample. small concentrations, especially those protein biomarkers that For example, researchers have demonstrated that use of nano- are very scarce in biological samples. The major drawbacks of cantilevers to detect specific DNA sequences may obviate the plasmonic ELISAs are their multi-step protocols and require- DNA-amplification step required by microcantilevers and other ment for trained personnel, making them best suited for labo- methods.[122] Nanocantilevers have already demonstrated good ratories and healthcare centers. However, it is envisaged its performance at determining antibiotic resistance in Escherichia integration along with smartphones and microfluidic devices coli.[119] Accordingly, nanocantilevers could be used to detect for its use in in-field measurements of human fluids such as nucleic acids that indicate microbiome-related skin condition urine, blood or plasma.[132–134] (e.g., atopic dermatitis), as they are present at only low concen- trations in skin-related samples.[124–126] However, since micro- cantilevers and nanocantilevers are very fragile structures, their 5. Future Perspectives portability is currently limited, thus precluding their use in PoC devices. Nevertheless, they can still be used as bench-top tools Here, we outline the three main features that we believe are in laboratories and healthcare centers. essential for any PoC sensor used for microbiome-related diag- nostics: multiplexing, smartphone connectivity, and automation (for sample processing). 4.2. Plasmonic Sensors Personalized medicine often entails the need to quantita- tively detect multiple biomarkers for a given disease. Mul- Plasmonic sensors based on plasmonic nanomaterials have tiplexing capability on a test is especially desirable for micro- been used in various biosensing strategies. Here, we present biome-related diseases, as mentioned above. In fact, depending two different types of plasmonic sensors: surface plasmon reso- on the relative abundance of each microorganism or biomarker nance (SPR) biosensors and plasmonic enzyme-linked immu- in question, different diagnosis might be reached (see, for nosorbent assays (ELISAs). example: Figures 2A and 2D). Furthermore, considering the Surface plasmon resonance is a physical phenomenon that ever-increasing number of microbiome-related biomarkers that occurs when free electrons (or surface plasmons) at the inter- are being discovered, we envision that nanobiosensors focused face of a conductive material and another medium oscillate on the microbiome could address multiple diseases and condi- when focused by polarized light. These oscillations are very tions within a single test. Researchers developing nanobiosen- sensitive to surface changes, making SPR sensors ideal for sors will have to face this problem and devise smart solutions label-free detection of molecular-binding events (e.g., antibody/ for the quantification of multiple biomarkers, possibly even of antigen binding). Nanomaterials such as AuNPs, AgNPs and different molecular classes. magnetic nanoparticles have been the most reported, mainly Widespread adoption of smartphones in developing and due to their high degree of modification and high refractive developed countries alike provides an ideal opportunity for PoC index.[127] For instance, Kaur et al. developed a highly sensi- devices based on nanobiosensors, either through direct integra- tive, highly specific ZnO/Au-based SPR biosensor for rapid tion or indirect coupling. Replacing complicated external equip- diagnosis of bacterial meningitis—a condition that can be ment with patients’ or caregivers’ phones would be a massive fatal if not diagnosed sufficiently early—based on detection of advance towards meeting the REASSURED criteria. Smart- Neisseria meningitidis DNA.[128] Similarly, Dey et al. developed phones enable real-time connectivity, are user-friendly and an SPR biosensor for detection and quantification of E. coli cells provide imaging and processing power sufficient for clinically in blood plasma.[129] Their device requires minimal operational relevant sensing performance. Interestingly, some PoC devices Adv. Mater. 2021, 33, 2006104 2006104 (10 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de (see, for example: Figure 2D) already include near-field com- assess any potential scientific, technical, ethical, legal, or soci- munication (NFC) technology, which enables users to exchange etal concerns.[143] data between a device and a smartphone. This is especially Human microbiome samples and their associated personal useful for monitoring, as it enables visualization of temporal data pose an ethical and legal challenge to protect individuals changes in biomarker levels and correlation of such changes and to preserve their privacy and the confidentiality. Thus, this with life events. Moreover, smartphones can convert qualitative area of research is covered by the European General Data Pro- measurements (e.g., those from LFAs or plasmonic biosensors) tection Regulation (GDPR) as well as by the Convention on Bio- into semi-quantitative measurements, and translate the com- medicine and Human Rights of the Council of Europe, which plex readout signals of multiplexed sensors into more compre- prohibits genetic discrimination.[144,145] Human microbiome hensible results (see Figure 2B).[132] samples should not be subject to due diligence obligations under Finally, given that detection of microbiome-related bio- EU Regulation 511/2014, as states have no sovereign rights over markers involves treatment of very small samples from diverse human microbiome samples or over their citizens.[146,147] Thus, matrices (e.g., blood, saliva, urine and stool), we feel that auto- the sole owners of these samples are the individuals (healthy mated processing of samples within diagnostic devices would volunteers or patients) from whom they were obtained. greatly facilitate PoC use. This can be done with lab-on-a-chip From a societal perspective, there is a need to raise aware- technology. For instance, samples can be sequentially treated ness about the value of human microbiome R&D, to educate (preconcentrated or purified) and sensed through the combined the public with accurate scientific evidence and to combat use of microfluidic channels with functional modules such misinformation. This in turn requires researchers to reflect as pumps, filters, actuators and sensors.[135–137] An example is on their priorities—namely, in terms of how the outcome of shown in Figure 3E, which depicts a tuberculosis diagnostics their research could benefit society at large. The general public device that detects Mycobacterium tuberculosis DNA in human should have a basic understanding of the importance of the sputum samples.[121] It comprises a PCR chamber for amplifi- human microbiome and of how to assess the quality of testing cation of sample DNA; mixing chambers; and a microcoil for and monitoring devices and services before accessing these nuclear magnetic resonance (NMR) measurements, equipped devices and services. with highly sensitive magnetic nanoparticles for enhanced NMR quantification. Microfluidics can also be applied for mon- itoring purposes. For instance, Koh et al. (Figure 2D) developed 6. Conclusions a wearable device for analysis of glucose, chloride, lactate and pH levels in sweat, which can be coupled to a smartphone.[75] Diagnosis of microbiome-related conditions through discovery, The technologies underpinning these examples could eventu- detection and measurement of appropriate biomarkers will ally be combined with one or more of the countless nanoma- be a cornerstone of future precision medicine. Here, we have terials now available, to provide PoC devices for continuous shown that the field of nanobiosensing offers ample opportu- monitoring of specific microbiome-related biomarkers. Such nities for future devices for such diagnostics, although many devices, when used by the patient at home, would make repeat challenges remain in engineering PoC devices that meet the visits to laboratories or healthcare centers unnecessary. How- REASSURED criteria. Each of the nanomaterials and nano- ever, automated sample processing for PoC devices continues biosensing technologies that we have covered here—including to be limited by reproducibility issues and the historic need for LFAs, electrochemical sensors, nanocantilevers, SPR biosen- on-board pumps. sors, plasmonic ELISAs and microfluidics—presents a unique combination of advantages and disadvantages for use in PoC devices. Furthermore, the quality of the newly discovered bio- 5.1 Ethical, Legal, and Societal Issues Pertaining to Human markers, and the analytical performance and robustness of Microbiome R&D and Innovation nanobiosensors regardless of underlying technology, all remain to be improved. We believe that the nanobiosensing commu- Growing debate over the human microbiome mirrors nity has a responsibility to patients to seize this opportunity by increasing promotion of personalized medicine, which prior- designing, developing and facilitating the commercialization of itizes R&D and innovation as well as projects mean to improve new nanobiosensors for microbiome-based diagnostics, espe- quality of life for citizens. Exploring the ethical, legal, and soci- cially for PoC applications. etal issues related to human microbiome R&D and innovation requires consideration of all pertinent local, national, European and International laws and guidelines. From the ethical and legal perspectives, human microbiome Acknowledgements samples should be treated as other human biological samples. The MICROB-PREDICT project has received funding from the European Thus, in general terms and with some exceptions, human Union’s Horizon 2020 Research & Innovation Program under grant microbiome samples could be used for research purposes but agreement No 825694. This article reflects only the authors’ view, and only upon informed consent of the owners (healthy volunteers the European Commission is not responsible for any use that may be made of the information contained therein. C.P. acknowledges the or patients). These considerations should also apply to regula- Marie Skłodowska-Curie Actions Individual Fellowship; this project has tion of biobanking of human microbiome samples. Accord- received funding from the European Union’s Horizon 2020 research ingly, human-microbiome R&D should be reviewed by inde- and innovation programme under the Marie Skłodowska-Curie grant pendent and multidisciplinary research ethics committees, to agreement No. 795635. Adv. 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Pelletier, D. Le Paslier, T. Yamada, [147] International Chamber of Commerce (ICC). Implications of EU D. R. Mende, G. R. Fernandes, J. Tap, T. Bruls, J. M. Batto, Regulation 511/2014 on Research on the Human Microbiome. Task M. Bertalan, N. Borruel, F. Casellas, L. Fernandez, L. Gautier, Force on Access and Benefit Sharing, December 2019. Celia Fuentes-Chust obtained her B.Sc. in biotechnology at University of Valencia in 2017 and an M.Sc. degree in advanced biotechnology at the Autonomous University of Barcelona in 2018. In 2018 she started working at the Nanobioelectronics & Biosensors group in the Catalan Institute of Nanoscience and Nanotechnology and started her Ph.D. in 2019 under the supervision of Dr. Claudio Parolo and Prof. Arben Merkoçi. Her work is focused on the development of lateral- flow paper-based point-of-care (PoC) nanobiosensors for microbiome-related diagnosis of acute-on-chronic liver failure (ACLF) and the development of systematic evolution of ligands by exponential enrichment (SELEX). Adv. Mater. 2021, 33, 2006104 2006104 (15 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Claudio Parolo obtained his Ph.D. in biotechnology from the Autonomous University of Barcelona in 2013 working on nanobiosensors. Since then he has focused his postdoctoral career on the development of point-of-care diagnostic tests. Specifically, he has worked on lateral flow assays (in Prof. McKendry group at the University College London) and electrochemical sen- sors based on nucleic acid scaffolds (in Prof. Plaxco group at the University of California Santa Barbara). In 2019 he joined Prof. Merkoçi’s group at the Catalan Institute of Nanoscience and Nanotechnology as Marie Skłodowska-Curie fellow working on the diagnosis of HIV and syphilis. Giulio Rosati is a postdoc researcher mainly interested on electrochemical biosensing with impedimetric and voltammetric techniques, on the development of inkjet-based fabrication platform for low-cost biosensors, and on the development of portable platform for their use at the point of care. Dr. Rosati received his Master’s degree in bioengineering in 2011 and a Ph.D. in biotechnology in 2015, both at the University of Padova, Italy. From 2015 he worked as a postdoc researcher in the BioDevices lab of the same university on projects in collaboration with several companies and spinoffs. Since 2019 he has been working in Arben Merkoçi’s group at ICN2, Barcelona. Lourdes Rivas carried out her chemistry studies at the Universidad Central de Venezuela (2007) and her Master’s degree in catalysis at the Universitat Rovira i Virgili in Tarragona (Spain) in 2009. She received her Ph.D. in chemistry at the Universitat Autònoma de Barcelona, under the supervision of Prof. Arben Merkoçi (2015) working in the improvement of nanoparticle-based biosensing platforms (electrochemical sensors and lateral flow assays). She continued as a post- doctoral researcher at the Kungliga Tekniska Högskolan (Sweden), Institute for Bioengineering of Catalonia, and currently, at the Catalan Institute of Nanoscience and Nanotechnology for the development of point of-care platforms for clinical applications. Karla Perez-Toralla obtained her Ph.D. in microfluidics from the Curie Institute and the Univer- sity of Paris, France, under the supervision of Prof. Jean-Louis Viovy (2013). She worked on the development of miniaturized devices for the detection circulating tumor biomarkers (University of Paris, 2016) and new surface functionalization strategies for the study of biological systems (University of Nebraska-Lincoln, USA, 2019), in collaboration with biologists and clinicians. She is now in charge of the microfluidics team at the Immunoanalysis Studies and Research Laboratory CEA-LERI, focused on the design and implementation of new diagnostic tests for the detection of pathogenic agents. Adv. Mater. 2021, 33, 2006104 2006104 (16 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Stéphanie Simon obtained her Ph.D. in 1999 and joined CEA in 2001. Since 2013, she has been managing the Immunoanalysis Studies and Research Laboratory (CEA-LERI), whose aims are the development and industrial transfer of immunological tests for the detection and diagnosis of biological threat agents and infectious diseases. As examples, an ELISA test for the diagnosis of bovine spongiform encephalopathy has been developed and commercialized by Bio-Rad in the 2000s, and more recently lateral flow immunoassays for the detection of bacterial antibiotic resis­tance have been transferred to NG-Biotech company and are now commercialized in more than 50 countries. Itziar de Lecuona obtained her M.A. in bioethics and law and received her Ph.D. in Law in 2011. She is Member of the UNESCO Chair in Bioethics and Deputy Director of the Bioethics and Law Observatory at the University of Barcelona (UB). She is member of the Consolidated Research Group “Bioethics, Law and Society” of the Catalonian Government, and member of the Catalo- nian Bioethics Committee (2015–2020) of the Research Ethics Committee at Hospital Clínic de Barcelona (2012–2020) and the Bioethics Commission at the UB. Recently, she became member of the Covid-19 mutlidisciplinary Working Group of the Ministry of Science and Innovation. Christophe Junot received his Doctor of Pharmacy diploma degree in 1997 and his Ph.D. degree in analytical chemistry in 2000. Then, he joined GlaxoSmithKline laboratories and was involved on pharmacokinetics and metabolism applied to drug discovery. Since 2002, he has been working at CEA on mass-spectrometry-based metabolomics for biomedical applications. He is now head of the Medicines and Healthcare Technologies Department, a joint unit between Paris-Saclay University, CEA, and INRAE, in which he coordinates research activities in the field of diagnostic and therapeutic innovation. He is also the deputy coordinator of MetaboHUB, the French infrastructure for metabolomics and fluxomics. Jonel Trebicka is Head of Translational Hepatology at the Department of Internal Medicine I at the Goethe University Frankfurt in Germany. He is also Research Coordinator in the European Foundation for Study of Chronic Liver Failure (EFCLIF) in Barcelona, Spain. He is coordinator of H2020-Project MICROB-PREDICT and principal investigator of the PREDICT study. He is associate editor of the Journal of Hepatology among others, as well as a member of the edito- rial board of Gastroenterology and Gut, among others. His field of interest and publications are basic and clinical research on end-stage liver diseases, HIV, and vascular liver diseases. Adv. Mater. 2021, 33, 2006104 2006104 (17 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH www.advancedsciencenews.com www.advmat.de Arben Merkoçi is ICREA Professor and director of the Nanobioelectronics & Biosensors Group at ICN2 in Barcelona. His research is focused on the design and application of cutting-edge nanotechnology and nanoscience-based cost/efficient biosensors. He has published around 300 peer review research papers, supervised 30 Ph.D. students and has been invited to give plenary lectures and keynote speeches in around 200 occasions in various countries. Prof. Merkoçi is Co-Editor In Chief of Biosensors and Bioelectronics and member of Editorial Board of other jour- nals. He is co-founder of two spin-off companies, PaperDrop dedicated to nanodiagnostics and GraphenicaLab to electronic printing. Adv. Mater. 2021, 33, 2006104 2006104 (18 of 18) © 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH