16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Review www.afm-journal.de Integrated Devices for Non-Invasive Diagnostics Hatice Ceren Ates, Anna Brunauer, Felix von Stetten, Gerald A. Urban, Firat Güder, Arben Merkoçi, Susanna Maria Früh, and Can Dincer* to access the dynamic physiological state “Sample-in-answer-out” type integrated diagnostic devices have been widely of the body and thus, improve patient’s recognized as the ultimate solution to simplify testing across healthcare sys- outcome. tems. Such systems are equipped with advanced fluidic, mechanical, chemical, These contemporary instruments biological, and electronic components to handle patient samples without deployed in specialized central laboratories any manual steps therefore have the potential to accelerate intervention and are not always accessible for continuous preventive monitoring; their utilization is improve patient outcomes. In this regard, the combination of integrated devices generally limited to reactive healthcare. and non-invasive sampling has gained a substantial interest to further improve Integrated devices aim to miniaturize the the comfort and safety of patients. In this Review, the pioneering developments analytical processes followed in the central in integrated diagnostics are covered and their potential in non-invasive sam- laboratories in a cost-effective manner by pling is discussed. The key properties of possible sample types are highlighted incorporating fluidic, electronic, mechan- ical, and chemical components within a by addressing their relevance for the clinical practice. Last, the factors affecting single unit. The philosophy of the inte- the transition of integrated devices from academia to the market are identified grated design aims to unify the sample by analyzing the technology readiness levels of selected examples and alterna- collection, preparation, analysis and post- tive remedies are explored to increase the rate of survival during this transition. processing to enable the “sample in– answer out” testing required for distributed proactive healthcare. In this regard, non- 1. Introduction invasive sampling offers an easy access to the physiologically relevant biomarkers with minimum discom- An ancient physician had to rely on perceptible observations fort and supervision. In the clinical practice, however, there are while examining a patient. This process might occasionally be many uncertainties regarding the diagnostic correspondence of accompanied by some ingenious tests such as the diagnosis of the measured concentrations in non-invasive samples and how diabetes mellitus from the attraction of black ants to the “honey these concentrations are correlated to the more familiar blood- urine”.[1] Since then, the diagnostic tools in the clinical practice based counterparts.[2,3] have extended well beyond the five human senses with the addi- In this review, we will assess the potential of eight different tion of advanced imaging techniques and chemical analysis of sample types (sweat, tear fluid, saliva, urine, breath, stool, bodily fluids. These new methods have strengthened the ability seminal fluid, and swab specimen) that could be collected H. C. Ates, Dr. C. Dincer Prof. F. von Stetten, Dr. S. M. Früh FIT – Freiburg Centre for Interactive Materials and Bioinspired Hahn-Schickard Technology Freiburg 79110, Germany University of Freiburg Prof. G. A. Urban Freiburg 79110, Germany FMF - Freiburg Materials Research Centre E-mail: dincer@imtek.de University of Freiburg H. C. Ates, Prof. G. A. Urban, Dr. C. Dincer Freiburg 79104, Germany Laboratory for Sensors Dr. F. Güder IMTEK – Department of Microsystems Engineering Department of Bioengineering University of Freiburg Imperial College London Freiburg 79110, Germany London SW7 2AZ, UK A. Brunauer, Prof. F. von Stetten, Dr. S. M. Früh Prof. A. Merkoçi Laboratory for MEMS Applications Catalan Institute of Nanoscience and Nanotechnology (ICN2) IMTEK – Department of Microsystems Engineering CSIC and The Barcelona Institute of Science and Technology University of Freiburg Barcelona 08193, Spain Freiburg 79110, Germany Prof. A. Merkoçi The ORCID identification number(s) for the author(s) of this article ICREA can be found under https://doi.org/10.1002/adfm.202010388. Barcelona 08010, Spain © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1002/adfm.202010388 Adv. Funct. Mater. 2021, 31, 2010388 2010388 (1 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de non-invasively and analyzed within the scope of integrated 2.2. Measurement Unit diagnostic devices. We will describe the composition (also known as “matrix”) and secretion/production mechanisms of The core of the integrated sensing platform is the measurement each sample type to explore their compatibilities with sample- unit, where the analytes interact with the recognition element. to-answer diagnostic methods. We will examine a series of diag- These interactions are mainly converted into an electrochem- nostic technologies at different technological maturities from ical or optical signal by the transducer, which is then traced by a the fundamental research phase to commercialization stage. detector unit. If necessary, transducer signals may be enhanced We will further discuss the factors influencing the translational by an amplifier before passing to the detector. success of “sample-to-answer” diagnostic devices by utilizing Recognition elements could be selected from a variety of the technology readiness level (TRL) concept and highlight the options with two distinct origins. Natural recognition ele- common pitfalls and possible remedies. ments are isolated directly from living organisms or bio- synthesized. The most frequently used bioreceptors include nucleic acids, enzymes, antibodies, membranes, bacterio- 2. Assembling an Integrated Device phage, organelles, and cells. Although they are highly specific and easy to produce at small scales, their mass production An integrated device is a platform that combines the funda- could be difficult and expensive at industrial scale. Further- mental building blocks of a process to convert the input variables more, they exhibit batch-to-batch variations, low stability, into a desired outcome within a self-confined environment. In and poor performance under non-physiological conditions. the case of point-of-care (POC) testing, an integrated diagnostic The other alternative is the artificial bioreceptors such as device may include different combinations of components for aptamers, molecularly imprinted polymers, supramolecular sample handling, recognition elements, signal processing and receptors, synthetic peptides and macrocycles.[4,6,7] Unlike transmission units, power supplier, and decision-making unit their natural alternatives, they are more compatible with (DMU). The extent of complexity of each unit as well their mass production, offer high affinity, and improved stability. assembly depends on the analyte of interest and the goal of Nevertheless, the development of a particular product is the measurement (qualitative/quantitative and single-shot/con- resource intensive requiring high capital investment and spe- tinuous), which in turn dictates the possible sample types to be cialized personnel. used for the desired task (blood, tear, saliva, urine, etc.). In the The next issue is the implementation of the selected recog- following, we will provide a holistic view on how this assembly nition elements and all related reagents into the measurement may be configured for different diagnostic purposes. unit. If the integrated platform is developed for multiple and/or continuous measurements, the supply of the key compounds must be ensured for the successful operation. For stationary 2.1. Material of Construction integrated platforms, auxiliary units for reagent storage, release, and manipulation can be included.[8–10] In this context, imple- There is no ultimate “all-in-one” material that can meet the mentation of microfluidics and automated flow control can fundamental requirements of various applications being help the realization of self-contained devices. However, such an developed under the integrated device concept, hence each approach is not practical for mobile or wearable configurations. specific task still requires a tailored material. In accordance, the In this case, label-free, wash-free strategies can be followed decision-making process starts with the selection of the “right” to bypass the need for delivery of reagents and buffers. As an construction material.[4] alternative, disposable measurement units may be utilized to The first input here is related to the selected sample type (tear, circumvent the regeneration issue.[7] urine, etc.) for the analyte (i.e., glucose) and its measurement The functionality of the measurement unit may also be strategy. The primary concerns differ significantly for a single- extended to multimodal/multiplexed sensing to improve the use, disposable device, and a platform for continuous moni- overall reliability of the system by i) reducing the false positives, toring. For example, while biocompatibility is a serious concern ii) providing multiple outputs to assess the physiological state, for a contact lens (CL) integrated glucose sensor, it is not a pri- and iii) providing the data for active calibration and correction. ority for a disposable, paper-based urinalysis for the same the This can be achieved by mounting different transducer types analyte. In the latter, choosing a biodegradable material is more and/or measuring multiple analytes on the same platform likely to become a design goal due to the environmental con- simultaneously. cerns.[5] In general, the physical requirements of a continuous monitoring platform are much more extended compared to disposable alternatives and must provide certain levels of flex- 2.3. Decision-Making Unit ibility and durability to be successfully integrated into the daily life of the users. Therefore, in addition to the analyte selection, The signal generated by the transducer(s) is converted into measurement strategy must be set priorly. The second step is to information in the DMU. Depending on the desired level of identify the possible techniques for the signal detection. Physical details (single/multi-measurements, single-shot/continuous, requirements of different methods would necessitate different/ qualitative/quantitative), DMU may consist of optical modules, additional properties from the sensing platform such as optical color charts and rulers, potentiostats, thermometric sensing access, stretchable electronics, and microfluidics. Based on devices, data storage and transmission units, integrated pro- these concerns and limitations, possible materials and the cor- cessors, and external auxiliary devices such as smartphones. responding manufacturing technologies should be outlined. If continuous monitoring is desired, the following questions Adv. Funct. Mater. 2021, 31, 2010388 2010388 (2 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de must be answered before finalizing the sheaves of sub-compo- 3.1. Sweat nents: i) Should the platform perform on-device-evaluation or raw/intermediate data is to be transferred out? ii) What should Sweat-based diagnostics have become a part of clinicians’ be the data acquisition and reporting frequency? iii) Is there a toolbox since the 1950s, which have recently gained a significant need for data storage? and iv) How is the power management momentum, particularly in the field of fitness monitoring. The handled? main driving force behind this attention is its rather convenient In the case of on-device-evaluation, transmitted signal(s) accessibility through widely distributed sweat glands across the must be processed, corrected, converted into meaningful infor- body, which makes it a suitable medium for integrated non- mation, displayed, and/or transmitted to the end-user. This invasive sensing. Nevertheless, its unique secretion mechanism policy would require an integrated decision unit (i.e., processor) still hinders the successful transition of sweat-based sensors while the burden on the data storage and transmission unit may into practice. The only noticeable clinical achievement is the be reduced. For external evaluation (smartwatch/phone, PC, cystic fibrosis (CF) screening.[15] cloud-based systems), the integrated device may follow a smart The sweat secreted by the eccrine glands carries the physi- data transmission scheme where the data is filtered, corrected ologically relevant biomarkers from blood to the accessible skin (cross-correlation) and converted. This approach would reduce surface for continuous monitoring via electroosmosis, which the volume data to be transferred yet also necessitates an inte- induces a pulsatile advective flow within the sweat duct. This grated decision unit. Alternatively, the raw (for example, current, discrete transport mechanism leads to a time lag and temporal voltage, light intensity) or intermediate (pH, concentration, tem- discontinuity for sweat-based analysis compared to blood/ perature) data can be transmitted without any diagnostic tags. interstitial fluid, hampering the possibility of instantaneous Data acquisition and reporting frequency strongly depend on monitoring. Another issue related to the analyte transport is the diagnostic role of the integrated platform. If the collected the presence of tight protein junctions along its path. These data is interpreted via benchmarking with the already existing proteins act like a transport barrier as they filter the large bio- databases and clinical models (such as diabetes, vitamin defi- markers and selectively reduce their concentrations along the ciency), volume of the transferred data may be reduced, as paracellular route. In other words, a correlation factor must be the decision-making process is fundamentally an “interpola- identified for each analyte to link the measured values with the tion” problem (i.e., where the individual measurement stands sweat gland concentrations in the dermis. In addition to this with respect to the population). If the integrated platform is passive filtering, analyte transport is also influenced by the lipo- designed with the scope of an individualized scheme, data philicity/hydrophobicity of the compounds as well as the pos- collection frequency must be sufficient enough to perform data- sibility of tight junction remodeling allowing the advection of driven regression analysis (i.e., how the analyte concentration large analytes.[16,17] This dynamic nature of sweat secretion is is changing with respect to the individual metabolism). A prac- not fully understood for most analytes and needs to be tackled tical example for the latter is the therapeutic drug management, before realizing the full potential of the sweat-based sensors. where the measured drug concentrations are processed to gen- This difficulty is reflected in the available studies and commer- erate an individualized pharmacokinetic/pharmacodynamics cial platforms (Figure 1), where the focus is usually on some fit- models for the patient.[11] Utilization of artificial intelligence (AI) ness indicators such as ion concentrations (sodium, potassium, and cloud-based data storage in this frame is becoming a virtual chloride) and rate of sweat loss, rather than the quantitative component of the integrated platforms in order to process large analysis of specific biomarkers for diagnostics. amount of data for pattern recognition and model abstraction.[12] Fitness monitoring avails of the wearable concept, which Another important component of the integrated platform is enables the continuous monitoring of various analytes in sweat the power unit, the size of which is typically determined by the via exercise-induced sampling. The underlying technology usu- DMU. Depending on the previous answers to the above-raised ally relies on electrochemical sensing where a printed circuit questions, the power demand of the integrated platform can board (sensing element) is integrated with a microfluidic patch. be estimated and a power supply strategy (simple storage, flex- In this ecole, multiplexed sensing is not uncommon and cur- ible batteries, solar or biofuel cells, hybrid systems[5,13,14]) must rent platforms are now offering quantitative and simultaneous be chosen. analysis of sweat ions, glucose, lactate, sweat rate, and physi- ological parameters such as pH, body temperature, and heart rate.[15,18–29] The alternative approach utilizes the optical sensing 3. Potential of Non-Invasive Sampling that can be deployed in the form of patches, integrable textiles, and tattoos.[2,16,17] Colorimetric analysis is also compatible in Integrated Devices with multiplexing and has been successfully implemented for Considering the opportunities offered by non-invasive sam- the detection of similar analytes.[30–33] Despite their quantita- pling to access the physiologically relevant biomarkers with tive nature, the overall focus of either approach is to provide minimum discomfort and supervision, in this section, we will information on the general state of the body, energy metabo- elaborate on the usage of eight potential sample types within lism, and the hydration state. For instance, quantitative glucose the frame of integrated diagnostic platforms. We will i) examine monitoring in this context refers to the analysis of diet and the analyte types and their transport mechanism in different exercise-induced changes and does not aim for a smart, non- samples, ii) discuss the operational challenges to process these invasive diabetes management. analytes, ii) assess the current state-of-the-art by going through Nevertheless, some important steps have been taken towards the recent literature. the utilization of sweat-based sensors (Figure 1) for the diagnostic Adv. Funct. Mater. 2021, 31, 2010388 2010388 (3 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 1. Integrated diagnostic devices for sweat analysis. A) Battery-free hybrid microfluidic device for electrochemical, colorimetric, and volumetric sweat analysis. Reproduced with permission.[26] Copyright 2019, American Association for the Advancement of Science. B) Laser engraved wearable device for uric acid and tyrosine detection for gout and metabolic disorders monitoring. Reproduced with permission.[36] Copyright 2020, Nature. C) Self-powered smartwatch for semi-quantitative continuous glucose monitoring. Reproduced with permission.[40] Copyright 2019, American Chemical Society. D) Skin- interfaced microfluidic system that can captures sweat rate and electrolyte concentration to provide personal sweat profile. Reproduced with permis- sion.[44] Copyright 2020, American Association for the Advancement of Science. E) Waterproof sweat patch for sweat collection, biomarker analysis, and thermography in aquatic settings. Reproduced with permission.[32] Copyright 2019, American Association for the Advancement of Science. F) Iontophoretic tattoo-based sensing for alcohol monitoring. Reproduced with permission.[45] Copyright 2019, American Chemical Society. G) Multifunctional microfluidic system for colorimetric measurement of sweat parameters and temperature. Reproduced with permission.[30] Copyright 2019, American Chemical Society. H) Smart ring can track the key signals from the body like heart rate, heart rate variability, temperature which has been recently offered by NBA to the players for “early illness detection monitoring” during coronavirus pandemic. Reproduced with permission.[21] Copyright 2020, OuraRing. applications.[23,27,30–42] In glucose monitoring, recent achieve- Sweat-based sensing platforms have made a significant ments include the increased capability of detecting small progress in device miniaturization, integration, multiplexing, variations in glucose concentrations after fasting/oral uptake,[43] signal processing, data transmission, and post-processing.[4,46] preliminary investigation of the blood-sweat correlation[27] and its In addition to the developments in the hardware design, there comparative analysis between the healthy and diabetic subjects.[39] are also important lessons learned through this experience, Another important progress is the development of integrated sen- which reveals the challenges originate from the inherent prop- sors for the CF diagnosis,[15,26,27,34] where the concept has been erties of sweat. One important observation is that the concen- shown to be in agreement with the conventional laboratory bio- tration of any analyte in sweat is influenced by internal and assays.[15] Gout monitoring is experiencing a similar evolutionary external factors such as sweat rate, body temperature, pH, pattern: gaining the ability to distinguish healthy individuals and the presence of other compounds. Therefore, accurate from the patients and correlating the sweat analyte concentra- and sensitive measurement of the analyte necessitates precise tions with those in serum.[36] Benchmarking with a gold standard (and additional) information about the instantaneous meas- method is another important milestone demonstrating the reli- urement landscape. As a result, multiplexed sensing becomes ability of an integrated platform, which has been, for instance, inevitable to represent these interdependencies accurately and recently achieved for continuous cortisol screening from sweat.[37] to capture the true physiological state of the body. Another Adv. Funct. Mater. 2021, 31, 2010388 2010388 (4 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de important deduction is related to the low secretion rates of conspicuous initiative on the integration of miniaturized elec- eccrine sweat, which usually dictates a need for sweat stimu- tronics with CLs for glucose sensing is the Google-Novartis part- lation. Fitness monitoring bypasses this challenge inherently nership initiated in 2014. Nevertheless, the project was halted while other diagnostic applications have to rely on chemical after 4 years due to the challenges experienced in accurate and or physical stimulation. Nevertheless, such attempts may reliable tear glucose measurements and insufficient consistency interfere with the secretion mechanism complicating the cor- between the blood-tear glucose concentrations.[49] Another focus relation problem or may simply create discomfort hindering of CL applications is the measurement of ocular pressure for the the success of application, which was the case for GlucoWatch, diagnosis of the glaucoma (Figure 2C), which has been recently an FDA-approved glucose monitoring device withdrawn from commercialized.[56] The device (SENSIMED Triggerfish) consists the market in 2007.[47] Above all, the nonlinear dependency of a telemetric CL sensor and an adhesive antenna wired to a between the sweat and blood concentration has to be estab- recorder, which operates similar to the Holter monitor. There are lished and clinically validated for any analyte of interest, before also other platforms in addition to CLs at various technological its appearance on the market as an end-use product. maturities including paper-based sensors for qualitative diag- nosis of dry eye disease,[57,58] microfluidic integrated eyeglasses (Figure 2D) for electrochemical detection of alcohol[59] and wear- 3.2. Tear Fluid able spring-like electrochemical sensor for glucose monitoring (Figure 2B).[60] A wide range of ions, big and small molecules can diffuse The difficulty in tear-based analysis is the scarcity of the basal into tear through the blood-tear barrier including proteins, tear, through which, in principle, a blood-tear correlation can be lipids, glycoproteins, hormones, electrolytes, nucleic acids, established. This constraint dictates the measurements to be per- and glucose. This diffusion process, in principle, underpins formed over the eye. Hence, both the sensing element and the the correlation between the blood and tear compositions. It data transmission unit must be kept in a delicate environment also acts like a transport barrier which eases the analysis of for extended periods of time. From an application point of view, it the sample by filtering the blood cells and reducing the matrix implies that all elements must be biocompatible, stable, energy- effect. This transport resistance, however, also decreases the efficient, stretchable, and miniaturized without compromising concentrations of analytes compared to their blood levels.[48,49] the sensitivity. Even this technical barrier is overcome, a greater The other major intricacy is related to the dynamic secretion quest is waiting to be challenged, that is, the in-vivo evaluation of mechanism of tears, which affects both the tear constituents the developed platform on human subjects, ideally including the and their concentrations. Basal tear fluid, which forms the per- validation studies for the tear-blood correlation. Finally, all these ennial protecting film covering the eye, is the most probable efforts should evolve into an affordable end-product. candidate for analyte monitoring as it is continuously in contact with the blood through the diffusion barrier. Nevertheless, the conventional approach (Schirmer strips) stimulates the reflex 3.3. Saliva tears during the sampling and inevitably change the analyte concentrations due to the differences between the compositions Saliva offers a direct but complex non-invasive path to measure and the secretion rates of basal and reflex tears. This difference the blood concentrations of many analytes that can be utilized further complicates the already unknown blood-tear correlation to trace the immunological state of a patient. Its complexity is by introducing another variable into the equation (reflex tear a result of its origin; a mixture of protein-rich secretions from flow rate and its composition).[11] The alternative is to collect the various glands (salivary, bronchial, and nasal), entrained epi- basal tears directly by using microcapillary tubes. In this case, thelial cells and microfauna, blood-derivatives due to gingival however, trained personnel are required to mitigate any cor- bleeding and exogenous biological, non-biological, organic, neal damage and the collectible sample volume is quite limited and inorganic impurities.[61] This intricate nature and the low (less than 5 µL).[1] Furthermore, neither of these approaches can concentrations of biomarkers are the Achilles’ heel of salivary be used for continuous, long-term disease monitoring such as diagnostics in a quantitative scheme. diabetes since they only allow discrete sampling. Presence of a biomarker, as well as its concentration, is Integrated sensing can answer the need for the long-term, strongly influenced by both the sample collection strategy continuous, on-site monitoring of basal tears, bypassing the (whether it is stimulated or not) and the sampling method difficulties of self-sampling and low-volume constraint. In this (passive drooling, spitting, swabs, dried saliva spots, or micro­ regard, utilization of CL is the “Columbus’ egg” of integrated sen- extraction). Furthermore, inherent properties of biomarkers sors, which are already adapted for daily life. CLs are easy-to-use, alter the stability of the compound in its native medium and disposable, and wearable for extended durations (due to the high pre-treatment of the sample may be required, such as the oxygen permeability).[50] The early examples of CL-based sensors addition of protease inhibitors to reduce protein degradation. were primarily focused on quantifying glucose in tear,[51,52] which Therefore, possible interferences and undesired side reactions is still a hot topic after two decades of fundamental research. The should be taken into account for each analyte during the pre- most recent developments include the possibility of multiplexed treatment, storage, and transportation steps. Integrated sensors, sensing,[53] utilization of advanced transparent soft materials and on the other hand, alleviate these challenges by introducing a wireless, real-time data transmission without an external power single-step, sample-in-answer-out approach, which has found source,[54] and integration of photonic nanostructures with com- its way into practice as either on-the-spot sensing platforms or mercial CLs for smartphone-assisted optical sensing.[55] One portable devices. Adv. Funct. Mater. 2021, 31, 2010388 2010388 (5 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 2. Integrated diagnostic devices for tear analysis. A) CL-based biosensor for continuous glucose monitoring. Reproduced with permission.[55] Copyright 2019, American Chemical Society. B) Spring-like device for electrochemical tear glucose monitoring. Reproduced with permission.[60] Copyright 2018, American Chemical Society. C) CL-assisted continuous ocular monitoring system for glaucoma guidance. Reproduced with permis- sion.[56] Copyright 2017, Dove Medical Press. D) Quantification of tear alcohol via electrochemical sensor mounted eyeglasses. Reproduced with permis- sion.[59] Copyright 2020, Elsevier. E) Multiplexed detection of tear biomarker via smartphone-assisted CL. Reproduced with permission.[52] Copyright 2020, Royal Society of Chemistry. F) Lateral flow cassette for dry eye disease diagnostics through elevated levels of inflammation marker (Matrix metal- loproteinase-9) in basal tear. Reproduced with permission.[58] Copyright 2020, Springer Nature. On-the-spot approach implies the utilization of wearables, commercially available test kits following this philosophy that which made their first appearance with mouth guards inte- are used for HIV,[75] drug abuse, and alcohol screening.[76] The grated with electrochemical sensors. One of the early examples other extreme is the use of natural sensors in mouth; that is is the real-time monitoring of uric acid in saliva (Figure 3E), the taste buds for chemical sensing. In this novel approach, the which can be used as a biomarker for the renal disorders.[62] chemical response of the taste buds to a particular analyte is Recently mouth guard applications for the measurement of exploited to create a qualitative analysis; for example, the expe- saliva glucose via well-established glucose oxidase reaction ena- rience of bitterness after chewing peptide sensor-loaded gum.[77] bling sensitive detection in the clinically relevant range have This approach may be holding the key in its design to estab- been reported.[63,64] A very similar design has also been utilized lish new saliva-based diagnostic tools, which can evolve towards for the diagnosis of degenerative cell changes such as Alzhei- “augmented sensing.” mer’s disease.[65] One recent study has followed an alternative Despite all the promising advances, there are still a number path by deploying a different sensing approach, which relies on of issues that are needed to be addressed.[11,61] The biggest chal- the colorimetric measurement using a disposable microfluidic lenge is to accurately identify the transport mechanisms. Ana- paper-based sensor.[66] Functionalized paper is placed into the lyte transfer is influenced by i) their unique physicochemical mouth guard, where the color change in glucose and nitride characteristics (lipophilicity, molecular weight, dissociation zones indicates the concentration of each analyte, respectively. constant, lipophilicity, pH, ionizability, and protein binding In addition to the mouth guards, there are also other on- affinity), ii) transport mechanism (active or passive), ii) sample the-spot sensing applications (Figure 3), where the integration collection method (degree of stimulation), iv) inter- and intra- notion is interpreted differently. Recent examples include i) individual variabilities, v) contamination (oral wounds, gingival glucose detection via toothbrush integrated non-enzymatic crevicular fluid, food/drink/smoking), vi) bias due to the oral sensor,[67] or ii) via printable organic thin-film transistors,[68] medications and proteolytic enzymes, and vii) degradation ii) tooth-mounted multiplexed sensor capable of measuring during the transport from blood to saliva. alcohol content, pH, temperature, salinity, and sugar level,[69] iv) pacifier-integrated enzyme-based electrochemical sensor for real-time health monitoring in infants[70] v) smartphone- 3.4. Urine assisted cortisol monitoring,[71] and vi) the dental retainer on which sodium intake is continuously measured by hybrid Utilization of urine as a diagnostic tool is as old as the recorded electronics.[72] history and it is still regarded as a versatile symptomatic tool for Portable integrated platforms are another alternative to the diabetes, bacterial infections, renal diseases, dehydration, preg- commonly used mouth guard approach, where the extrac- nancy even genitourinary cancer. The urine formation process tion, delivery, and measurement of the collected analyte are provides sufficient residence time for the diffusion of metabo- handled via an external all-in-one platform.[74] There are some lites, salts, proteins, and other valuable analytes from nearby Adv. Funct. Mater. 2021, 31, 2010388 2010388 (6 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 3. Integrated diagnostic devices for oral fluid analysis. A) Portable diagnostic device for electrochemical salivary cotinine detection. Reproduced with permission.[73] Copyright 2019, Wiley. B) Mouth guard biosensor for in vivo glucose measurement. Reproduced with permission.[64] Copyright 2020, American Chemical Society. C) Wearable “in-mouth” platform for electrochemical sodium intake monitoring toward hypertension management. Reproduced with permission.[72] Copyright 2018, PNAS. D) Continuous uric acid detection via mount guard biosensor with integrated wireless elec- tronics. Reproduced with permission.[62] Copyright 2015, Elsevier. E) Pacifier biosensor for continuous measurement of salivary biomarkers in infants. Reproduced with permission.[70] Copyright 2019, American Chemical Society. F) Smart toothbrush for glucose monitoring via electrocatalytic oxidation. Reproduced with permission.[67] Copyright 2019, Elsevier. G) Smartphone-assisted LFA for chemiluminescence-based cortisol detection. Reproduced with permission.[71] Copyright 2015, Elsevier. blood capillaries, which in turn creates a practical means to test strip into a smart toilet design,[83] which makes real-time, measure biomarker concentrations reflective of the patient cloud-based urine analysis possible. The toilet is also equipped status. As in the case of other non-invasive mediums, this with a uroflowmetry, capable of identifying the abnormal urine diffusion process leads to much lower analyte concentrations, flow rates associated with diseases. hence only those having high plasma levels become detectable Commercially available integrated sensors cover a broad in urine samples unless highly sensitive detection methods are range of applications from immunochromatographic test strips employed. with reference color charts to sophisticated reader-integrated Majority of the integrated devices developed for urinalysis devices, which are now being utilized to monitor infectious dis- utilizes paper-based sensors (Figure 4), which in turn rely eases, cancer, diabetes, and kidney diseases. Dipstick assays are on optical sensing where the signal readout is performed via the most popular and well recognized paper-based commercial color reference charts, optical readers, or handheld devices. urine analysis strategy, where the pregnancy tests can be exem- In this regard, integration of the smartphones to sensors has plified as the most established ones.[84] They are also utilized brought new possibilities thanks to their enhanced imaging for the analysis of urine chemistry via measuring several key and computing capabilities, prevalent existence in the market, parameters including glucose, bilirubin, ketones, specific and familiarity for the end-users as well as the technology gravity, and pH.[1,85] Several companies (Abbot, Polymedco, SD developers.[78] Recent examples of this perspective are a hybrid Biosensor, NephroCheck, Quidel) have introduced a variety of device for the quantification of urine analytes,[79] the detection immunochromatography-based lateral flow assays (LFAs) to of bacterial infection via “fidget spinner” like lab-on-a-disk the market for qualitative and rapid analysis of urine for the platform,[80] and a fluorescence microarray for the detection diagnosis of infectious diseases, tuberculosis, sexually trans- of breast cancer-specific biomarkers.[81] An alternative route to mitted diseases (STDs), bladder cancer, acute kidney injury, eliminate the need for an external readout for the quantitative and sepsis.[1] These products, however, may lead to the misjudg- analysis is the utilization of an integrated scale (for example, a ment of test results due to the variations in color interpretation ruler), as illustrated for the simultaneous detection of glucose by the end-users. In order to alleviate this problem, companies and bovine serum albumin.[82] Another interesting paper-based have launched various kinds of optical readers like Roche Urisys sensor study is the integration of an already available urinalysis or NephroCheck Astute140 Meter.[1] Another problem related Adv. Funct. Mater. 2021, 31, 2010388 2010388 (7 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 4. Integrated diagnostic devices for urine and stool analysis. A) Screen-printed electrodes for simultaneous detection of electroactive bio- molecules (ascorbic acid, dopamine, and uric acid) in urine. Reproduced with permission.[87] Copyright 2018, Elsevier. B) POC testing of urinary tract infections through analysis of bacterial culture via centrifugation-based fidget spinner. Reproduced with permission.[80] Copyright 2020, Nature. C) Lateral flow pregnancy test strip. Reproduced with permission (https://en.wikipedia.org/wiki/Clearblue). Copyright 2013, SPD Swiss Precision Diag- nostics GmbH. D) Lateral flow cassette for diagnosis and monitoring of inflammatory bowel disease by the detection of calprotectin in stool samples. Reproduced with permission.[88] Copyright 2020, Nature. E) A “smart toilet” system for urinalysis via monitoring of urine analytes (proteins, glucose, ketones, hemoglobin, bilirubin, urobilinogen, acetone, nitrite, and leucocytes) and urine flow patterns. Reproduced with permission.[83] Copyright 2020, Nature. F) Electrochemical DNA biosensor for Salmonella detection from stool samples. Reproduced with permission.[89] Copyright 2018, MDPI. with the immunochromatographic assays is their low sensi- the healthcare system. This limited access to the laboratories tivity. Commercial fluorescent-based products, in this respect, creates another issue with respect to the stool handling. The have gained popularity as a solution to the sensitivity problem, sample itself is biohazardous, which necessitates special care especially for acute kidney injury detection.[1,86] during the sampling and its transportation. Furthermore, the time delay between the sample collection and analysis may influ- ence the test outcomes depending on the analyte. For instance, 3.5. Stool the viability of the intestinal bacteria within the sample is prone to changes depending on the sample handling[91] while the Stool is commonly tested in medical practice particularly for durability of a simpler analyte such as hemoglobin is found to be the disorders of the digestive system. Its significant potential fairly stable.[92] Therefore, development of integrated devices is of for integrated sensors is associated with its rich constituents significant importance for the realization of POC stool analysis. including many biomarkers, cells, bacteria, viruses, fungi, and The use of microfluidics, which is one of the fundamental other parasites. Stool cultures are used for the diagnosis of building blocks of integrated sensor designs, has the poten- pathogenic diseases such as dysentery as well as various cancer tial to overcome these challenges. Previous examples of types. Besides, gut microbiota is considered to be correlated microfluidic devices in this context include on-chip detection with neurodegenerative diseases like Alzheimer’s disease.[90] of antigens, bacteria, and nucleic acids.[93–95] Nevertheless, Compared to other non-invasive methods, conventional most of these achievements rely on off-chip sample preparation stool analysis requires complex sample processing, sophisti- steps such as vortexing, filtration, and centrifugation, hence cated equipment, and well-trained personnel. Its labor-inten- hampering the evolution of conventional methods to portable sive and time-consuming nature also reduces its availability in “sample-in-answer-out” devices. Adv. Funct. Mater. 2021, 31, 2010388 2010388 (8 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de A promising step towards this goal is the developments in be measured for a given disease and establishing the method on-chip sample processing. One recent example is the direct of detection for the selected analyte. As the first step, exten- processing of crude stool samples by using a microfluidic sive effort has been put into the analysis of the biomarkers in system for Helicobacter pylori detection. The sample is first breath, which reveals some key VOCs that are strongly cor- mixed with magnetic beads, which extracts the DNA from the related to certain metabolic activities. In this regard, breath bacteria. In the next step, beads are transferred into an elution analysis has been performed particularly for the diagnosis of chamber where the DNA is purified for further analysis.[96] diabetes and lung cancer. For diabetes, acetone concentration In another study, stool samples are homogenized by using in breath is monitored, which is typically found to be at least acoustic streaming created in the microfluidic device. Its two times higher than that of a healthy person. Lung cancer unique design enables a gentle, continuous flow keeping the monitoring relies on the ratio of certain VOCs[98] rather than cells intact, which is an important criterion for downstream their concentrations due to the fact that changes in metabolic applications such as flow cytometry, cell detection, and culture. activities lead to detectable variations in these defined ratios. A fully-automated integrated microfluidic device has also been With respect to its design philosophy, the preferred method of recently presented for Salmonella detection in stool (Figure 4F). detection in breath sensors has been inspired by nature; that is It deploys antibody-coated gold nanoparticles to identify mimicking the sense of smell. The basic principle behind the bacterial DNA by using electrochemical sensing.[89] The only smelling is the detection of trace amount of VOCs entrained completely integrated paper-based device for stool analysis is an within the air via neuron cell receptors and the interpretation aptasensor, which uses two separated reaction zones connected of the generated signal in the brain. Similar to the receptor by a diffusion bridge enabling the detection of different bacte- cells present in the nose, breath sensors also deploy a variety of rial markers in spiked stool samples.[97] chemical receptors, which is combined with a data acquisition There is a limited number of low-cost commercial system and a database for the decision-making process. Such applications for integrated stool analysis, mainly due to the systems are also referred to as the electronic nose (e-nose) tech- challenging sample preparation step. One particular example is nology in the literature.[99,100] the Calprotectin Home Testing system (Figure 4D) developed by One well-recognized commercially available e-nose example Bühlmann Laboratories AG, which measures the fecal calpro- is Cyranose 320, which deploys an array of nanocomposite sen- tectin concentration via smartphone application for the diag- sors for the “smell print” detection. It has been successfully nosis of inflammatory bowel disease.[88] A completely different tested for obstructive airway diseases such as asthma, CF, lung perspective is the smart toilet concept, which has been around cancer, infectious and inflammatory diseases.[101] The Aeonose, since the mid-1980s. One recent study has presented a proof-of- another commercially available e-nose application, processes concept toilet technology for continuous health monitoring.[83] exhaled breath directly without using any temporary storage In this system, stool images are automatically taken and graded unit for the analysis of lung cancer-related VOCs.[98] One according to Bristol stool scale with a machine learning algo- study worth noting here is the recently developed UV-irradi- rithm, which can correlate the data with the user via biometric ated e-nose application,[102] which can distinguish between the identifiers. Total seating and stool dropping times are also breath patterns of lung cancer patients from the healthy sub- processed for monitoring the constipation and hemorrhoids. jects. Furthermore, it can also discriminate the type of cancer In its current state, it is not capable of biochemical analysis of as well as its major subtypes (squamous cell carcinoma and stool, yet claimed to be implemented in the near future. adenocarcinoma). In addition to these e-nose applications, there are also studies utilizing different detection mechanisms for breath analysis 3.6. Breath (Figure 5). One interesting example is the miniaturization of the gas chromatography for the detection of lung cancer- Exhaled breath, with more than 3000 volatile organic com- associated VOCs in breath.[103] The proposed system integrates pounds (VOCs) identified in its content, holds potential for microelectromechanical systems with metal oxide semicon- POC diagnostics. In particular, endogenous VOCs are of ductor technology, which includes a separation column, on-chip significant importance from a clinical point of view as they are detector, and signal processing unit. Recently, a multiplexed, the by-products of metabolic activity. These trace molecules are nanomaterial-based sensor array has been demonstrated for carried by the blood circulation and diffused into the exhaled the detection of COVID-19-related VOCs. Initial results dem- breath through respiratory fluid within the lungs, which creates onstrate that proposed device can successfully differentiate the possibility to correlate the blood and breath concentra- patients from control groups, as well as COVID-19 from other tion levels for a given compound. Exogenous VOCs, on the lung-related diseases.[104] other hand, are originated from the external sources hence Another recent example utilizing a different design phi- their concentrations in breath provide valuable information losophy is the paper-based wearable humidity sensor for about the environmental exposures, drug intake, and patient’s breath analysis.[105] The working principle of the device relies habits. Nevertheless, presence of these external substances on the change in the ionic conductivity with the water content may hinder the measurement of the metabolically relevant absorbed in the paper, which makes it possible to deploy the compounds, which are typically at very low concentrations device as a portable humidity sensor to track the real-time res- (1–5000 parts-per-billion).[98] piration. A similar approach has also been demonstrated as a Utilization of sensors for diagnostics can be interpreted proof-of-concept technology for monitoring lung and infectious as a two-step design process: deciding on which analyte is to diseases[106] through the electrochemical detection of hydrogen Adv. Funct. Mater. 2021, 31, 2010388 2010388 (9 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 5. Integrated diagnostic devices for breath analysis. A) Wearable paper-based sensor for continuous respiration activity monitoring. Reproduced with permission.[105] Copyright 2016, Wiley. B) Nano-material based multiplexed sensor array for the detection of COVID-19-related VOCs. Reproduced with permission.[104] Copyright 2020, American Chemical Society. C) Internet-of-things-enabled e-nose for breath analysis. Reproduced with permis- sion.[99] Copyright 2020, MDPI. D) Wearable paper-based sensor for electrochemical analysis of different analytes (such as hydrogen peroxide) in exhaled breath. Reproduced with permission.[107] Copyright 2019, American Chemical Society. E) UV-irradiated e-nose for prediction of lung cancer histological types through exhaled breath. Reproduced with permission.[102] Copyright 2020, Elsevier. F) Wearable e-nose for real-time health status through monitoring of armpit odor. Reproduced with permission.[100] Copyright 2014, MDPI. peroxide from exhaled breath.[107] The paper-based device com- More importantly, there is still no consensus in the community prises two geometrically identical electrodes (Figure 5D). The on how to interpret the measured biomarker levels in breath first electrode measures the background signals caused by quantitatively, as reliable correlations between breath-blood interfering substances or environmental conditions (like tem- concentrations are still not well-established. perature and humidity), while the sensing electrode is specially sensitized to detect the target molecule. Breath sampling is performed on pre-treated cellulose paper, which acts like both 3.7. Seminal Fluid a substrate (i.e., a carrier for electrodes and/or biomolecules like enzymes or antibodies) and a solid electrolyte for the Semen contains male gametes (sperm) in a cocktail of various electrochemical detection. These two examples indicate that proteins, enzymes, vitamins, and other substances such as the paper-based approaches can offer a promising solution for sugar, which provides a nutrient media for the cells. Its analysis accessible, simple, and wearable breath testing by integrating forms the base of male fertility tests and relies on the quanti- sampling, collection, and analysis steps into a single device. fication of its physical properties (pH, color, viscosity) as well The challenges in breath analysis are all stemmed from the as the sperm deficiencies including its number density, vitality, low biomarker concentrations. Instantaneous concentration movement ability (motility), DNA integrity, and morphology. of those trace molecules strongly depends on the subject (age, Semen testing is also important for the diagnosis of STDs. sex, diet, medicine, fasting, smoking) as well as the expiratory Conventional clinical methods include counting chambers, flow rate. Furthermore, breath sample can be contaminated computer-assisted sperm analysis (CASA), viability, and integ- by the presence of exogenous VOCs. The composition can be rity assays. Nevertheless, these methods involve complex and also influenced by the sample collection method, for instance, time-consuming procedures requiring expensive equipment the possible contamination due to the interactions with saliva. and skilled personnel. In this regard, integrated devices offer Adv. Funct. Mater. 2021, 31, 2010388 2010388 (10 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 6. Integrated diagnostic devices for swab sample and seminal fluid analysis. A) Disposable nucleic acid amplification platform for the detec- tion of MRSA bacteria genes from nasal swabs. Reproduced with permission.[112] Copyright 2016, Royal Society of Chemistry. B) 2D paper network for the detection of the influenza from nasal swab samples. Reproduced with permission.[113] Copyright 2019, Royal Society of Chemistry. C) Lateral flow home sperm count test. Reproduced with permission.[114] Copyright 2010, Oxford Journals. D) Centrifugal PCR-based microfluidic device for detection of infectious diseases. Reproduced with permission.[115] Copyright 2020, Spindiag. E) An automated smartphone-assisted device for semen analysis. Reproduced with permission.[108] Copyright 2017, American Association for the Advancement of Science. F) Nucleic acid amplification assay integrated with a smartphone-based well plate fluorescence reader. Reproduced with permission.[116] Copyright 2018, American Chemical Society. a remedy to overcome these drawbacks as they offer simple, FertilMARQ, SpermCheck, and SwimCount can be listed as the rapid, and disposable solutions, which also enhances the pos- commercially available examples of this approach. Another sibility of on-site diagnostics. Home testing, in particular, has qualitative analysis method uses centrifugal forces for the the potential to encourage compliance among male patients by sperm count, available in the market under the brand Trak. reducing the anxiety and discomfort during the sampling. Similar to the staining-based methods, it only indicates the There are successful implementations of integrated sensors sperm concentration within wide ranges, and further testing for the semen analysis, which incorporates already available is still required to determine the abnormalities in sperm mor- smartphone capabilities for optical sensing and data analysis. phology and motility.[110] One example utilizes a capillary-based negative pressure Irrespective of the method deployed for the semen anal- feeding system to analyze unprocessed, crude semen samples ysis, collection, and delivery of the samples might play a sig- to quantify sperm concentration, and motility (Figure 6E). The nificant role in the results. Accordingly, best practice guide platform has been tested by benchmarking its prediction with prepared by WHO should be followed for the sample collec- those of CASA and showed a good agreement.[108] Very recently, tion: i) The sample should be collected where the test will be this smartphone-based semen analyzer has been further performed to minimize the temperature fluctuations and to improved with new capabilities for measuring sperm viability, standardize the sample-to-result time, ii) sample should be binding, and DNA fragmentation.[109] collected after a minimum 2 days and a maximum of 7 days Most of the current commercial home-based male infer- of sexual abstinence, ii) Semen sample needs to be complete tility assays, on the other hand, only measure the sperm con- any loss of the semen sample fraction must be reported, iv) centration. Chemical staining of the proteins on the sperm sample container should be clean, wide-mouthed, made of head is one of the commonly used methods for that purpose, glass or plastic which is confirmed to be non-toxic for sper- which demonstrates qualitatively whether the sample has matozoa, v) specimen should be kept at the temperature higher or lower sperm concentration than a threshold value. range of 20–37 °C.[111] Adv. Funct. Mater. 2021, 31, 2010388 2010388 (11 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de 3.8. Swab Specimen nucleoproteins for the diagnosis of infectious diseases, by using either fluorescent immunoassays or immunochromatographic Swab sampling is a common non-invasive and economic diag- tests. However, they only provide qualitative results unless an nostic method for various kinds of diseases including cervical external readout unit is deployed and the sample preparation cancer, influenza, and lung infections.[112,113,116–121] It can be col- has to be handled by the end-user. lected from the nose, pharynx, throat, urethra, cervix, vagina, wounds, and rectum. Proper specimen collection is the most critical step in swab analysis, which consists of sterilization, 4. Translation to the Market site preparation, swabbing, and transportation. Depending on the site of application, the swab itself must ensure the right Transition of an idea to an end-product is an evolutionary pro- flexibility, shape, and size while the tip material must have cess. Each technology must survive through various phases the appropriate properties enabling efficient sample capture with its own success criteria and risks until it reaches the last and target release. This type of an approach, when combined stage, commercialization. Technology readiness assessment with an integrated sensor, has the potential to improve the is a formal evaluation method developed to navigate through management of epidemic outbreaks (such as COVID-19) as it this landscape while minimizing the associated risks at each offers the possibility of early diagnosis owing to its on-site, fast, level. This is commonly achieved by tracking the developing affordable, and disposable nature. idea via TRLs tailored for different fields such as biomedical One of the earliest examples of integrated paper-based applications. devices for swab samples was developed for the early diag­­­­­ TRL concept was first introduced by NASA as a means to nosis of influenza from nasal samples[121] where results were assess the maturity of new technologies for risk manage- compared with those obtained with a gold standard method. ment and technology transition, particularly for the defense The first disposable nucleic acid amplification platform is also industry.[126] The current scale consists of nine levels spanning developed for the detection of bacterial gene targets from nasal from the observation of basic principles to the production and swab samples.[112] In this paper-based design, two methicillin- distribution stage. The purpose of the assessment is to reflect resistant Staphylococcus aureus (MRSA) related genes are the scale of the application (component, subsystem, system) monitored for the diagnosis of infectious diseases (Figure 6A). and the applied environment (laboratory, mathematical and Design objective of this particular system is to develop an physical simulations, field tests, and operational use) hence affordable disposable sensor to be used in resource-limited does not indicate the difficulty in achieving the next level or the areas. A centrifugal microfluidic LabDisk device has recently associated risks. Nevertheless, the method has proved itself as been introduced for the on-site and rapid detection of MRSA, a convenient tool for navigating the technology landscape and which improves the disease screening and timely decision- further adopted for biomedical systems. making process.[122] The proposed design also increases The TRLs for pharmaceuticals and medical devices follow the the antibiotic therapy efficiency via clear differentiation of same main frame of the original classification which is tailored methicillin-sensitive and resistive bacteria. by the FDA regulations and previous experience of the medical Another paper-based nucleic acid amplification system is industry (Figure 7).[126] These modifications reflect the three char- reported for the early diagnosis of influenza from clinical naso- acteristics observed in medical technologies: i) risk assessment, pharyngeal specimens.[123] The extraction, amplification, and ii) technological evolution, and ii) flexibility. In product develop- detection of viral RNA are handled on the same chip, within the ment, the risk associated with the new technology is normally clinically-relevant detection limit. The same group also introduced expected to decrease with increasing TRL. For medical applica- a solution for the early diagnosis of sexually transmitted infec- tions, however, the risk reduction does not change linearly across tions from vaginal and urethral swab samples.[124] An internal the levels and the risks remain high until the very late stages. control system is added to the design enabling the differentiation A medical device does not necessarily follow a sequential path of negative test results from invalid measurements, which during its technological evolution either and it is not uncommon further increases the reliability for POC applications. to see the change to be greater than a single TRL (for instance, A recently emerging trend is to combine LFA with CRISPR from TRL 4 to TRL 8). Therefore, the formal classification should technology for various infectious diseases,[117–120] as it provides be interpreted as a general guideline for the routine sequential low-cost, simple, and easily programmable tools for sensi- developments. In accordance, the line passing through the grey tive and selective detection of nucleic acids. The versatility of area between the successive TRL decisions becomes inherently CRISPR-associated (Cas) enzymes enables i) to target a variety flexible and should be drawn by considering the maturation and of nucleic acids (i.e., double-stranded DNAs or single-stranded risk characteristics of a particular technology. RNAs or DNAs), ii) to work under different cleavage conditions When we applied the TRL scale to the recent non-invasive (temperature or buffer), and ii) to combine analytical proce- integrated platforms (Table S1, Supporting Information), three dures such as sample extraction, amplification, and analysis features were frequently observed: i) current studies are une- in a single step.[117] In accordance, CRISPR/Cas technology venly distributed among four major categories: research phase, can enhance the evolution of the existing biosensing devices integration phase, near-commercialization, and commercializa- towards “sample in-answer out” solutions. tion (Table 1); ii) there exists a vast pool of fundamental research There are also commercially available kits for the analysis (TRL 1–2) which is intrinsically reflected as high number of avail- of throat, nasal or nasopharyngeal swab samples.[1,115,125] These able laboratory models (TRL 3); ii) the success rate of reaching sensors are capable of detecting bacterial antigens or viral TRL 7 and beyond is quite low. Here the critical question is what Adv. Funct. Mater. 2021, 31, 2010388 2010388 (12 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Figure 7. TRL scale for diagnostic devices. inherent characteristics of a particular application increase the developing technology. An integrated device could be designed rate of survival during the integration phase. TRL distributions as a supportive tool easing the decision-making process for in terms of sample type indicate that there is no apparent cor- the physician. The other extreme is to build the device with an relation between the matrix type and the success rate. However, integrated decision unit so that unsupervised testing is possible there exists a significant segregation with respect to the ana- for all end-users. This distinction results in different priorities lyte of interest, its complexity, and the sensing technology. The regarding various components, where the survival of the tech- majority of the commercialized platforms focus on biomarkers nology depends on investing in the right subsystems. Incorpo- whose presence or absence is sufficient enough to describe the rating the environmental concerns into the design philosophy medical status, rather than the case where its precise concentra- may also affect the long-term survival of a device in the market. tion is needed to be monitored continuously in a dynamically Utilization of single-use products such as pregnancy, ovulation, changing environment (for example, glucose in sweat). Another or infertility tests has been raising environmental concerns; important observation is related to the sensing strategy. Com- therefore, a clear distinction between reusable and disposable mercialized examples aim to answer “yes/no questions” with parts of an integrated device should be made during the product qualitative results, which is usually achieved via LFAs. There- development. A successful example of responding to such con- fore, individual challenges of each matrix type for the quanti- cerns is a recently developed biodegradable, flushable pregnancy tative analysis such as stability, cross-contamination, transient test which eliminates the plastic waste created by the disposable nature of analyte concentrations, data transmission, and energy counterparts.[127] Another factor improving the success rate of an supply have not been addressed and rather bypassed. integrated device is its familiarity and comfort for the end-users. Market expectations and the selection of a target group are In this regard, the current trends combine sensors with smart- expected to play a significant role in the successful transition of phones, CLs, and wristwatches, which will ease their integration Adv. Funct. Mater. 2021, 31, 2010388 2010388 (13 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de Table 1. TRL assessment of selected integrated devices for non-invasive diagnostics. TRL Matrix Analyte Target Technology Ref. 3 Stool DNA Salmonellosis Electrochemical [89] Urine Ascorbic acid, dopamine, uric acid Urinary tract function Smartphone-assisted electrochemical [87] Swab Hemagglutinin Influenza LFA [113] 4 Oral fluid Sodium Hypertension Electrochemical [72] Oral fluid Glucose and nitride Diabetes mellitus Colorimetric μPAD [66] Sweat Glucose Wellness monitoring Electrochemical [40] Sweat Glucose, lactate, choline Wellness monitoring Electrochemical [42] 5 Semen Concentration & motility Infertility Smartphone-assisted optical [108] Breath Humidity Sleep apnea Electrochemical μPAD [105] Swab DNA Bacterial infection LFA [112] Oral fluid Cotinine Tobacco exposure Electrochemical [74] 6 Breath VOCs Lung cancer Electronic nose [102] Oral fluid Glucose Diabetes mellitus Electrochemical [68] Urine Urine biochemistry Urinary tract function LFA [83] Sweat Uric acid, tyrosine Gout disease Electrochemical [36] 7 Urine Bacteria Urinary tract infection Colorimetric [80] Sweat Sweat rate, analyte concentration Sweat profile Hybrid [32] 8 Breath VOCs Obstructive lung disease, cancer Electronic nose [128] Breath VOCs Infectious diseases Hybrid [104] Tears Glucose Diabetes Electrochemical [60] 9 Tears MMP-9 Dry eye disease LFA [58] Swab Antigen or protein Infectious disease LFA [125] Sweat Sweat rate, analyte concentration Sweat profile Colorimetric [44] Urine Urine biochemistry Urinary tract function LFA [129] Urine hCG Ovulation/pregnancy LFA [84] Stool Calprotectin IBD LFA [88] Semen Concentration Infertility LFA [114] to our daily life. One striking example concerning the signifi- analysis answering some basic “yes/no” questions while the cance of comfort for the end-user is the short commercial life of success rate of quantitative continuous monitoring is notice- GlucoWatch, which is partly due to the complaints about long ably low. The first major obstacle hampering this transition is warm-up times, painful rashes, and skin irritation. the low concentrations of physiologically relevant analytes in non-invasive matrices, that is, the concentration of the analyte of interest compared to the native components constituting the 5. Outlook matrix is extremely small. Detection in such challenging ratios is analogous to counting the footsteps of a bug behind a railway The essence of the integrated sensing platforms is the “sample track while a train is passing.[130] Accordingly, the success of the in-answer out” approach which has the potential to provide developed device is essentially tied to the sensitivity and accu- real-time physiological data via miniaturizing the complex racy of the selected method. Therefore, this design bottleneck laboratory procedures into one small device. The phrase should be alleviated by either improving the currently available “sample in” implies that the sample should be easily accessible alternatives or introducing a new approach, while ensuring and collectible without any expertise or external devices, compatibility with the rest of the components of the integrated making the non-invasive sampling a suitable choice. Neverthe- platform such as data transmission and power supply units. less, this approach has still not reached its anticipated potential Even this technical barrier is overcome, the data interpretation and has not paved its way into practice. step still awaits as a greater challenge. The link between the Our analysis reveals that the variations in the technological instantaneous blood analyte concentrations and those meas- maturity of non-invasive integrated devices are independent ured in the non-invasive matrices is still missing. This physio- of the matrix type and rather depends on the sensing strategy. logical correlation gets further complicated due to the inherent Applications survived the basic research step and successfully dependence between the sample collection method and the gained their places in the market usually rely on qualitative analyte concentrations.[11] Our progress in understanding this Adv. Funct. Mater. 2021, 31, 2010388 2010388 (14 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de complex puzzle is directly scaled to the physiological insights Keywords deduced from the measured concentration profiles. Thus, a sig- integrated sensors, microfluidics, non-invasive diagnostics, point-of-care nificant amount of resources must be allocated to identify the testing, wearables details of these transport mechanisms starting from individual to wider populations. 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Advanced Functional Materials published by Wiley-VCH GmbH 16163028, 2021, 15, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202010388 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.afm-journal.de [128] Canary Health, Decoding the data in your breath, https://www. will-the-noninvasive-glucose-monitoring-revolution-ever-ar- canaryhealthtech.com (accessed: August 2020). rive-30754 (accessed: May 2020). [129] Roche, Combur Test strip, https://www.roche.de/diagnostik- [131] D. Silver, J. Schrittwieser, K. Simonyan, I. Antonoglou, produkte/produktkatalog/tests-parameter/combur-5-test-hc A. Huang, A. Guez, T. Hubert, L. Baker, M. Lai, A. Bolton, (accessed: August 2020). Y. Chen, T. Lillicrap, F. Hui, L. Sifre, G. Van Den [130] C. Offord, Will the Noninvasive Glucose Monitoring Revolu- Driessche, T. Graepel, D. Hassabis, Nature 2017, 550, tion Ever Arrive? https://www.the-scientist.com/news-analysis/ 354. H. Ceren Ates received her B.Sc. and M.Sc. degrees in Chemical Engineering and Micro and Nanotechnology Departments from Middle East Technical University, Turkey, in 2015 and 2018, respectively. She has also worked as a clinical research R&D engineer in Cellsway (Mikro Biyosistemler Inc.) during 2015–2018. Ceren is currently a scientific researcher at Freiburg Center for Interactive Materials and Bioinspired Technologies (FIT) and pursuing her Ph.D. degree in microsystem engineering at University of Freiburg. Her area of expertise is the development of bioanalytical devices for on-site diagnostics and therapeutic drug monitoring. Can Dincer is currently junior research group leader at the Freiburg Center for Interactive Materials and Bioinspired Technologies (FIT) of the University of Freiburg. Having completed his studies in microsystems engineering, he received his Ph.D. degree with summa cum laude in microsystems engineering from the University of Freiburg. Between June 2017–June 2019, Can also worked as a visiting researcher at the Department of Bioengineering at the Imperial College London. The main research interest of his working group “Disposable Microsystems” is the devel- opment of bioanalytical microsystems for various applications including diagnostics, especially wearables and POC diagnostics, food, and environmental monitoring. Adv. Funct. Mater. 2021, 31, 2010388 2010388 (18 of 18) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH