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MEMS biosensors and COVID-19 : missed opportunity
Thierry Leichlé, Liviu Nicu, and Thomas Alava
ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.0c01463 • Publication Date (Web): 25 Sep 2020
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MEMS biosensors and COVID-19 : missed
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opportunity
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16 Thierry Leïchlé1,2*, Liviu Nicu2, Thomas Alava3
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19 1GeorgiaTech-CNRS
20 Joint International Laboratory, School of Electrical and Computer
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22 Engineering, Atlanta, GA, USA
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25 2LAAS-CNRS, Université de Toulouse, CNRS, Toulouse, France
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28 3Université
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Grenoble Alpes, CEA, LETI, Grenoble, France
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35 KEYWORDS: MEMS, BioMEMS, biosensors, COVID-19, SARS-CoV-2
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42 ABSTRACT
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46 The acceleration of climatic, digital and health challenges is testing scientific communities.
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48 Scientists must provide concrete answers in terms of technological solutions to a society which
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expects immediate returns on the public investment. We are living such a scenario on a global
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53 scale with the pandemic crisis of COVID-19 where expectations for virological and serological
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55 diagnosis tests have been and are still gigantic. In this article, we focus on a class of biosensors
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60 ACS Paragon Plus Environment
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ACS Sensors Page 2 of 27
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3 (mechanical biosensors) which are ubiquitous in the literature in the form of high performance,
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6 sensitive, selective, low cost biological analysis systems. The spectacular development announced
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8 in their performances in the last 20 years suggested the possibility of finding these mechanical
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10 sensors on the front line of the COVID-19 but the reality was quite different. We analyze the cause
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of this rendez-vous manqué, the operational criteria that kept these biosensors away from the field
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15 and we indicate the pitfalls to avoid in the future in the development of all types of biosensors of
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17 which the ultimate goal is to be immediately operational for the intended application.
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24 In 2008, two of the authors of this article published a review article presumptuously titled
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26 “Biosensors and tools for surface functionalization from the macro- to the nanoscale: The way
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29 forward”1. The aim of the article was to present a global view of the functionalization and
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31 transduction techniques used in the field of biodetection as well as to provide a perspective in this
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33 same field for a new category of biosensors (new at that time), namely that of nanobiosensors.
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36 Being researchers from the micro and nanoelectromechanical systems (M(N)EMS) community,
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38 the emphasis was on mechanical transducers. The last two sentences or the article’s conclusion
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40 were: “Finally, is it the fact that having such a tremendous choice of systems would sometimes
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complicate the choice itself [of the right biosensor for a given application]? This is another
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45 question that remains to be solved”.
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47 Twelve years later, in the midst of the COVID-19 pandemic, these two sentences resonate as a
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49 premonition. Indeed, among the plethora of biosensors and functionalization methods described in
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52 the review, none has been the subject of a viable alternative to the gold-standards techniques such
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54 as PCR (Polymerase Chain Reaction) and ELISA (Enzyme Linked ImmunoSorbent Assay) to
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3 carry out virological or immunological tests for the detection of viral antigens and antiviral
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6 antibodies (informing about a current or past infection)2. However, while the successively affected
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8 countries were confining their populations one after the other from the end of January 2020 (first
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10 China, followed by Italy, France, Spain, the UK…) and as we were witnessing the shortage of
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conventional tests and reagents necessary to make them work, the scientific community mobilized
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15 to seek to implement new testing methods able to identify sick people and monitor the spread of
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17 the virus – identified as one of eight research action priorities by the World Health Organization
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(WHO)3. As scientists in the biological microelectromechanical systems (bioMEMS) community,
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22 we had two immediate reflexes: (1) look through our own arsenal of microdevices for the available
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24 technological bricks to create an operational system; (2) send a call to friends and colleagues in
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26 our community to elicit the same reaction and try to get an immediate operational response. These
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29 two actions produced the same result, namely the inability to deliver any viable technical solution.
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31 Once the excitement of the moment was overcome, we faced the reality that principally
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33 molecular diagnosis tools such as real-time reverse transcriptase PCR (qRT-PCR), and to a lesser
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extent – since not recommended by the WHO for clinical decision-making4 – ELISA and LFA
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38 (Lateral Flow Assays) were going to definitively lead the battle5 (each technique with its limits,
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40 well known and assumed). And we quickly witnessed the approval and the commercialization of
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kits for COVID-19 diagnostic6,7, but none relying on MEMS or other micro and nanotechnology-
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45 based sensors. Hence, we resolved to the idea that we had to carry out a retrospective analysis of
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47 the rendez-vous manqué between the mechanical biosensors and the actual historical pandemic.
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49 If we analyze the bibliography during the last 20 years, we observe that despite the “pandemic”
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52 growth of papers related to “mechanical biosensors” (almost 1,700 % growth, with more than 160
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3 journal papers published last yeari), none of the systems described as ultimate tools has been able
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6 to provide a viable solution to the needs of virological and immunological tests for clinical
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8 diagnosis and surveillance or to the fundamental questions related to the mechanisms of the SARS-
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10 CoV-2 infection.
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The main questions are: why this failure? Why, despite more than 20 years of research in this
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15 field involving universities from 65 different countries and relying on funding from more than 220
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17 different funding agenciesii, no mechanical biosensor (whatever its level of maturity) opens any
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prospect in terms of response to the needs arising from the current health crisis?
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22 The purpose of this article is to open the horizon beyond the field specific to bioM(N)EMS and
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24 to invite similar communities to avoid the pitfall of too much confidence and self-sufficiency by
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26 offering turnkey solutions that have no reality suited to the real world. This will save time, money
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29 and will allow digging other, much more promising leads that we will attempt to outline at the end
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31 of the article.
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53 i Web of Science analysis
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3 The process of molecular analysis: general considerations, specific context of SARS-CoV-2 and
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6 how BioMEMS happened to be unfit for the real world
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8 In medical biology, molecular analysis is carried out following specific steps, from sample
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10 collection to the interpretation of the results. The analytical procedure can be broken down in three
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elements8. First of all, the analytical principle that concerns the measurement or detection
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15 technique and that applied physicists and engineers call the biosensor. A biosensor consists of a
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17 bioreceptor coupled to a transducer that translates a biorecognition event into a measurable signal,
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where MEMS is a class of mechanical transducers9. Secondly, the analytical method which
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22 includes the sample preparation steps used to optimize the conditions suitable for detection (i.e.
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24 adapted to the detection technique), as well as the technological means implemented for the correct
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26 operation of the sensor (e.g. fluidics, temperature control, measurement electronics, user interface,
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29 etc.): this corresponds to the instrument. Finally, the complete analytical procedure that covers the
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31 entire measurement chain, from sampling to the final information.
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33 The steps of collecting and interpreting the results are the responsibility of medical professionals:
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qualified personnel collect samples following specific protocols to ensure validity and conformity,
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38 and only a medical doctor is ultimately authorized to issue a diagnosis based on the results of the
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40 analysis. This is the procedure followed in medical analysis laboratories equipped with high-
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performance measuring instruments (that have benefited from major technological advances since
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45 the 1960s and today offer the possibility of carrying out precise and exhaustive analyses on a large
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47 number of samples with a quality approach that leads to minimized errors10). This is not the case
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49 for point-of-care (POC) tools or bedside devices that are divided into two sub-categories11: bench-
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52 top analyzers, which are ultimately miniaturized versions of conventional systems, and portable
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54 devices, such as immuno-chromatographic tests on strips/LFA or in-vivo sensors. For example,
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3 diabetics now have access to analytical tools for monitoring their blood sugar levels and in this
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6 context, take their own sample – a drop of blood obtained by pricking one's finger12. They are then
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8 able to interpret the results of the measurement and make decisions on their diet. Of course, this
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10 example cannot be transposed to more complex pathologies at the moment, but it is the most
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emblematic case of the bedside analytical tool, as opposed to centralized analysis platforms. Still,
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15 the main reason for the use of POC tools is the speed of turnaround and response, which is crucial,
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17 for example, to determine in emergency departments through cardiac biomarker assays whether a
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patient with acute chest pain is having a myocardial infarction or, in the present COVID-19 crisis,
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22 if a patient with flu-like symptoms should self-quarantine because of infectious risks.
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24 Before getting further into the discussion of key elements in biosensing, especially in the context
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26 of the current COVID-19 outbreak, let us argue about what went wrong with the application of
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29 bioMEMS as virological and serological tools on the battle field against the SARS-CoV-2 virus.
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31 BioMEMS can be defined as electro-mechanical devices or systems fabricated using
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33 micro/nanoscale technologies and dedicated to the analysis/sensing/identification of specific
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biological entities or the interaction in between them. If we consider that areas of research and
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38 applications of bioMEMS range from microfluidics, surface functionalization to tissue
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40 engineering, implantable microsystems, etc., then bioMEMS have been designed, developed and
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promoted for more than twenty years as the ultimate tools for rapid, low-cost, ultra-sensitive
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45 diagnosis of all kind of pathologies that are affecting the human being. And all this, not to mention
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47 their nanoscale counterparts, the bioN(ano)EMS, which ultimately have been advertised as the
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49 future nanosytems for complex biosensing13, meaning that for such a basic challenge of
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52 quantifying the viral load of a known virus (SARS-CoV-2) or the amount of antibodies anti-SARS-
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3 CoV-2 in a patient serum, bioNEMS would have done the job more easily and brightly than
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6 standard tools such as PCR or ELISA.
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8 In the last two decades, the conviction that BioM(N)EMS, and especially resonators, of all shape
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10 and size, were to revolutionize the market of biological detection was strong. Seminal papers were
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published to demonstrate the nano-mechanical resonators potential to ultra-high sensitivity to mass
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15 loading14–16. Simultaneously, complementary metal oxide semi-conductor (CMOS) integration of
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17 MEMS/NEMS resonators and parallel addressing of thousands of NEMS became feasible17.
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Finally, a lot of works demonstrated the ability of attaching aptamers18, antibodies19, enzymes20,
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22 and DNA probes21 to the surface of cantilevers to specifically capture biological targets of interest
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24 – including viruses22 – within a sampled liquid volume.
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26 Every single specification from high sensitivity (generally more often very low limit-of-
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29 detection), specificity to target, massive multiplexing (multiplexed electrical/optical addressing)
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31 has been demonstrated and a road to a handheld, on-field biological testing unit relying on the
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33 prowess of M(N)EMS seemed imminent. Moreover, with numerous companies already developing
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and selling physical sensors that include MEMS solutions, we were expecting to see MEMS-based
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38 devices invading the market of point-of-care biological sensing. Even if we have witnessed the
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40 successful use of MEMS sensors for health monitoring such as CardioMEMS, the very first
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implanted biomedical MEMS sensor for monitoring the pulmonary arterial pressure developed by
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45 Mark Allen in the early 2000 at Georgia Tech, and which is now a device recommended for
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47 patients with of heart failure in Europe23, these physical sensors are not, by definition, biosensors.
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49 Hence, the only evidence we can observe is that the invasion of MEMS biosensors has not
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52 happened yet and the next paragraphs will provide part of the explanation for such broken
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54 promises.
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3 The overwhelming majority of point-of-care tests actually performed on the field on a daily basis
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6 are of two types: 1 – fast response time tests that can provide results in a matter of minutes with
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8 moderate sensitivity and mostly consisting of rapid diagnostic strip tests24 and, 2 – ultrasensitive
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10 molecular tests in which sampling is done on the field but analysis (that requires chemical and
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primers) is led in a lab setting and provide results within a few hours. One could argue that
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15 molecular tests, such as PCR, are not performed “on field”, yet, in developed countries, the
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17 network of laboratories that can perform theses analysis can be sufficiently meshed so that the
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travel to these facilities can be integrated in the total analysis time. Moreover, with the advent of
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22 isothermal amplification techniques (e.g. Loop-Mediated Isothermal Amplification, Clustered
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24 Regularly Interspaced Short Palindromic Repeats-triggered amplification), several scientific teams
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26 are now able to bring the entirety of the chain of analysis on the field with microfluidic lab on chip
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29 devices and lateral flow readouts that prove efficient in reducing the analysis response time25–31.
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31 Both rapid tests and molecular diagnosis tools have the monopoly in answering the need for on
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33 field biological tests with respectively very different features and technical means. They have been
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able to do so because they have the figure of merit that mostly matters for the users: confidence in
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38 the result. For authorities and healthcare operators relying on these tests to cope with important
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40 decisions, the advent of false positive or false negative tests could lead to critical decisions such
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as population lockdown whereas there is no need or vice-versa. Applying aggressive counter-
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45 measures to a situation falsely identified as sanitary-threatening can have dire consequences.
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47 MEMS biosensors (here shortly and abusively called bioMEMS) consist of two parts: a means
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49 of transduction of the biological event happening on the active surface and a layer of biologically
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52 specific recognition molecules; thus, their development is intrinsically pluridisciplinary.
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54 BioMEMS is currently pushed by scientific experts from the field of micro and nanotechnologies.
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3 Immunologists, molecular biologists and functionalization experts have still little involvement in
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6 the development of bioMEMS solution. As a result, one first key mistake is that functionalization
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8 of bioMEMS is often put last, using standard (and too often inadequate) techniques and paying too
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10 little attention on its implementation. Obviously the transducer is the means to ultralow mass or
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charge sensitivity and can detect a small handful of biological species but the biological receptors
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15 layer held to the transducer is the actual interaction space between the biological targets in its
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17 medium and the sensors itself. It is natural to consider that one needs to better understand the
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mechanics of bioreceptor-target interaction at the sensor’s active surface level to increase the
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22 confidence level in BioMEMS.
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24 A second key mistake when developing biological total analysis systems based on BioMEMS with
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26 the intent of transferring this technology to end users lies in poorly identifying the figure of merits
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29 the final system needs to compel to ensure acceptance by the users. BioMEMS scientific teams
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31 usually center most of the developments on performances such as ultralow limits of sensing,
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33 massive multiplexing and miniaturization. Despite ultralow mass sensitivity for resonant
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bioMEMS, as a matter of fact already demonstrated for virus detection32, or massive multiplexing
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38 of the bioNEMS functionalization33, these techniques will fail to convince end users if they do not
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40 take into account the issue of the robustness of the provided detection answer. Ultimately, micro
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and nanotechnological bricks for biological analysis will not be considered as alternative
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45 technologies to the conventional gold standards on the field for users will prefer the latter ones as
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47 best providers regarding the confidence in the final result and the ability to answer real needs of
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49 specific biosensing applications (Fig. 1), as it will be discussed in the following sections.
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30 Figure 1. “The smoke and mirrors of mechanical biosensors” illustrates the gap between the
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32 questions researchers and end-users are aiming to answer.
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37 Biosensors
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40 Microelectronics-derived technologies for molecular analysis
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42 The expected contribution of technologies derived from microelectronics (i.e. microsystems,
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44 microfluidics, nanotechnologies) to the field of molecular analysis is twofold: first, the
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miniaturization of systems and the possible reduction of device costs, and second, the development
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49 of new detection principles to improve sensitivities and lower detection thresholds.
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51 The vast majority of biosensors, which transform a biological recognition into a measurable
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physical signal, are based on electrochemical and optical transductions34, but many other
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56 approaches have also been proposed, including ones calling for electrical and mechanical, e.g.
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3 MEMS, transducers. Thus, the literature on this subject is comprehensive and even if very few
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6 products have made it to the market with regard to the volume of research (we can nevertheless
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8 cite the Surface Plasmon Resonance (SPR), Quartz Crystal Microbalance (QCM) and other digital
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10 PCR technologies), the expected impact of these technologies on the world of molecular analysis
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is tremendous. However, in order to have a real medical utility, and this is especially true to fight
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15 the COVID-19 pandemic the world is now facing, the specifications to be met are extremely
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17 demanding and there are still many challenges to overcome.
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22 What role for the biosensor: screening/diagnosis/prognosis/disease management?
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24 Molecular assays that look for specific biomarkers can have several clinical uses. Thus, the first
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26 question that should be asked when developing or wishing to use a biosensor in the biomedical
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29 field is: for what purposes and in which cases should it be used? Indeed, even if in the introductions
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31 of publications presenting detection platforms, the application is often fairly well tailored in terms
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33 of biomarkers to be detected and pathologies to be treated, the role of the biosensor is often less
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well defined.
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38 Pendley and Linder have recently discussed the role of the biosensor from the end-user (i.e. the
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40 physician) perspective35. What emerges from their study is that the appropriateness of using a
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43 biosensor depends less on its performance than on the prevalence of the target disease. Indeed,
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45 because the sensitivity and specificity of a sensor (i.e. the ability to appropriately deliver true
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47 positive and true negative results) cannot be 100%, the use of a sensor to diagnose a low-
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prevalence disease in a large population seems unrealistic and useless (the authors take the example
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52 of influenza diagnosis during and outside of epidemic periods). Worse still concerning screening:
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54 it is, for example, vain to imagine being able to conduct a routine screening test for pancreatic
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3 cancer without having multiple false positives to treat (and in this case, what should be
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6 communicated to the patient?). If, on the other hand, the test is restricted to a population at risk or
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8 during a pandemic, then the analysis obtained by the sensor will be more useful: it is obviously the
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10 case of sensors developed to monitor the spread of COVID-19. More specifically, while high
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sensitivity tests (with low false negative) are mandatory for properly diagnosing COVID-19 cases,
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15 surveillance of the population requires kits with high specificity (i.e. low false negative)6. If former
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17 tests can be carried out in central laboratories, it is highly desirable to have access to low cost POC
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devices for surveillance means.
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22 Whatever the purpose of the biosensor, it is crucial to consider the context of the analysis as
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24 much as the performance of the instrument. In the case of biomarkers that are highly diluted in the
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26 samples to be analyzed, such as circulating DNA in oncology, the analysis is likely to be more
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29 limited by the method of sample collection than by the performance of the detection method. The
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31 sampling location and sampling time is also of upmost importance, as illustrated by the variability
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33 of viral loads of the SARS-CoV-2 in respiratory samples (throat and nasal swabs, sputum samples),
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36 urine, and stool36,37. Additionally, the sampling time, which in the case of the COVID-19 translates
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38 to the number of days after infection, provides a snapshot of the stage of the disease and is thus
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40 also a crucial parameter to take into account when choosing the test type: while virological tests
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43 seek a current infection, immunological tests will prove more useful days after the infection to
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45 look for antibodies produced against the virus38. Similarly, while it is obvious that the detection
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47 limit of a sensor is one of its main characteristics that is often the first consideration, as long as
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this detection limit is sufficient to detect the lowest level of analyte necessary for a clinical decision
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52 with minimum uncertainty, it does not need to be optimized. Furthermore, for this characteristic
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54 to be relevant, it must have been measured under real conditions, i.e. on the final sample (e.g.
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3 detection in serum and not in buffer). Unfortunately, articles presenting detection methods often
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6 report detection limit values without justification of the method and calculation used for their
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8 determination.
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A double requirement : sensitivity and specificity
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15 Considering the level of concentration of some biomarkers in liquid biopsies, which is below ~
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17 3 × 103 copies/mL for a positive diagnosis of SARS-CoV-2 by qRT-PCR assay39, it is obvious
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20 that there is a need for highly sensitive biosensors for many clinical applications. Sensors based
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22 on new technologies display very high sensitivities, although the decrease in active surface area
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24 and lower concentrations are also accompanied by a longer analysis time40. It should also be noted
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27 that the measurement of very low concentrations involves analyzing volumes large enough to
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29 minimize statistical errors due to sampling and to work within a comfortable confidence interval41.
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The specificity of a biosensor determines its ability to avoid false positives. It is therefore a
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34 crucial characteristic. It is the bioreceptor layer, due to the affinity of the probe molecules used for
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36 the targets, that provides the specificity of the biosensor. In complex samples such as liquid
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38 biopsies, the presence of many molecules can interfere with the measurement by non-specific
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41 interactions with the sensor. The concentration of many plasma proteins is several orders of
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43 magnitude higher than that of protein biomarkers42. Hence, even if the affinity of the interfering
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45 molecules is very low, they are the source of a strong biological background, which can exceed
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48 the nM13. The consequence of this background noise is that it drastically reduces the effective
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50 sensitivity of the sensor, as has been shown during the determination of miRNAs by hybridization
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using microcantilevers in the presence of total RNAs43. It is therefore important to minimize the
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3 influence of this biological background noise, either by differential measurement or by suitable
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6 sample preparation.
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10 Analytical method
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Point-of-care liquid biopsy analyses: desired characteristics
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15 The term liquid biopsy is used to refer to a test performed on a body fluid as opposed to a
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17 conventional biopsy, which consists of removing a small piece of organ or tissue. Liquid biopsy,
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and particularly blood or saliva sampling, can ultimately be considered the most widely used
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22 sample format in molecular analysis. The determination of biomarkers in liquid biopsies using
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24 sensors at the patient's bedside is particularly demanding: the ideal sensor would thus have the
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26 characteristics of being at the same time portable, cheap, robust, fast, sensitive, specific,
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29 multiplexed (i.e. allowing multi-analyte determination) and requiring a very small volume for
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31 analysis44; the order of importance of these criteria varies, of course, depending on the
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application45.
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38 Role and importance of the pre-analytical sample preparation
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Sample preparation is an essential step in the analytical process that makes the sample
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43 compatible and optimized to a given measurement technique. The functions commonly used to
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45 perform sample preparation include sample purification and suspension (removing interfering
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47 species and dissolving the sample in a suitable solvent by filtration and extraction separation
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50 techniques), sample concentration (increasing the local concentration of analytes lowers the
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52 detection limit of the analytical technique) and sample modification (using e.g. derivatization,
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54 amplification, cell lysis, or enzymatic digestion)46.
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3 These steps commonly involve centrifugation, phase separation, and extraction kits. They
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6 generally lead to dilution of the sample, which is obviously not favorable when low concentration
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8 biomarkers are to be detected. Most of the sample preparation steps are not automated and the
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10 results are highly dependent on the lab technician and the followed protocol. In addition, it has
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been shown that despite the use of commercial products, the wide variability in their performance
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15 – such as for example the yield rate of miRNA extraction kits that is not always consistent47 – has
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17 a tremendous influence on the analytical results. Hence, pre-analytical steps, including sample
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20 preparation, are recognized as the predominant source of errors in laboratory medicine10. As a
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22 result, the need to improve pre-analytical procedures has been highlighted in a number of studies
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24 on diagnostic methods48.
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26
27 From a biosensor point of view, it is essential to detail and take into account the sample
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29 preparation procedures in order to correctly assess the performance of an analytical technique,
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31 especially with regard to analysis time and sensitivity. Besides, in order for a miniaturized analysis
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33
34 technique to be as efficient as possible – in the sense that it must bring significant improvements
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36 over existing techniques (and obviously in terms of portability or overall assay time), it is important
37
38 to minimize the on-bench steps of sample preparation. While one strategy is to develop analytical
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41 techniques that require minimal sample preparation, another strategy is to integrate on the chip as
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43 many of these steps as possible within the biosensor’s instrument. Numerous technological
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45 solutions for integrating specific pre-analytical functions on a chip can now be found in the
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47
literature, even if sample preparation can still be considered a real obstacle to the deployment of
48
49
50 POC biosensors49.
51
52 Finally, it is obvious that the preparation must be adapted to the analytical technique. The latter
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imposes tight specifications, more or less restrictive, in terms of sample format and composition.
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15
1
2
3 For example, the amplification of nucleic acids by PCR requires the presence of numerous
4
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6 biomolecules in solution (primers, DNA polymerases, mixture of the four deoxyribonucleotides),
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8 direct electrical detection on silicon nanowires can only be carried out in low ionic strength buffers
9
10 that display a fairly large Debye length50 and dosing with mechanical microresonators is largely
11
12
13 influenced by the viscosity of the solution51. In this respect, since physiological liquids have
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15 viscosities close to that of water, the latter MEMS technology is particularly suitable for the
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17
analysis of liquid biopsies with minimal sample preparation, as demonstrated by Raj Mutharasan's
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19
20 team for the direct determination of miRNA biomarkers in serum52. Thus, among the solutions
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22 offered by new technologies, some biosensors offer undeniable advantages with regard to pre-
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24
analytical requirements and are therefore more or less likely to provide truly nomadic analytical
25
26
27 instruments.
28
29
30
31 Analytical process
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34 Portability and cost
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36 Works dealing with biosensors based on micro and nanotechnologies always emphasize the
37
38 benefits of integration and cost reduction. As a matter of fact, MEMS physical sensors such as
39
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41 accelerometers and pressure sensors commercialized by Bosch, ST Microelectronics or Analog
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43 Devices are as small and lightweight as packaged integrated circuits and are very cheap, typically
44
45 a few USD, because they are mass produced: it turns out that the current need for COVID-19 tests,
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47
especially for surveillance means, corresponds to the large volume of sensor units required to
48
49
50 achieve a low cost per unit. While integration capability can indeed lead to portable solutions, it
51
52 should be noted that unlike the sensor itself, the instrument is much more complicated to
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54 miniaturize53. Fortunately, microfluidics now provides answers to miniaturization through lab-on-
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16
1
2
3 a-chip approaches54 but there are many other factors that still limit the deployment of on-field
4
5
6 operational sensors for biomarker dosing, such as biocompatibility and robustness of the device or
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8 the reliability and stability of the measurement.
9
10 Furthermore, it is important to note that the significance of these criteria is relative and depends
11
12
13 solely on the application. For example, the cost of a disposable sensor for the diagnosis of
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15 infectious diseases in high-risk areas is a decisive criterion and must obviously be as low as
16
17 possible, which is not necessarily the case for a sensor used in a hospital environment to
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20 accompany cancer treatment55: same applies to the fight against COVID-19 where, while it is
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22 highly desirable to have disposable low-cost devices to massively test the population in public
23
24 spaces, such as airports, the cost of tools to backup computed tomography (CT) scans for SARS-
25
26
27 CoV-2 diagnosis on a selected population in a hospital might not be that critical56. From this
28
29 example, other characteristics and technological choices can be discussed according to the
30
31 application context57.
32
33
34
35
36 The importance of multiplexed analysis
37
38
The determination of PSA levels in blood has been used since the 1980s for the early detection
39
40
41 of prostate cancer before the appearance of clinical signs. However, an elevated PSA level is not
42
43 specific for cancer but simply marks the presence of an abnormality in the prostate. Thus, the
44
45 diagnosis must be confirmed or disproved by further tests or clinical examinations. While this test
46
47
48 is of great value given the prevalence of prostate cancer and the slow progression of the disease, it
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50 is now clear that the use of multiple biomarkers provides more accurate information about the
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52 presence and stage of a cancer. The CancerSEEK test introduced in 201858 perfectly illustrates this
53
54
55 point: an assay of 8 circulating proteins and the search for mutations on circulating tumour DNA
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17
1
2
3 from 61 amplicons proved to be able to diagnose the presence of cancer (among 8 types of cancer)
4
5
6 with a sensitivity above 70% and a specificity above 99% (on a sample of 1005 patients). In the
7
8 frame work of the COVID-19 pandemic, genetic tests require the analysis of a number of viral
9
10 genome sequences (e.g. the RdRP, E and N genes) to identify the 2019-n CoV and discriminate it
11
12
13 from other coronaviruses, such as MERS-CoV or SARS-CoV59.
14
15 Being able to detect several molecules on the same platform is an absolute necessity in practice
16
17 in order to provide at least one reference and to take the biological background noise into
18
19
20 consideration. Ideally, it would also be appropriate to have a means of calibrating the sensor by
21
22 dosing molecules present in the sample at steady and known concentrations (so-called
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24 "housekeeping molecules"60, such as the gene coding for the human Ribonuclease P used as a
25
26
27 reference in RT-PCR for SARS-CoV-2 detection61). Multiplexing poses real technological
28
29 challenges: in addition to making multiple sensors on the same chip, it is necessary to be able to
30
31 interrogate them individually and deal with the disparity in response sensitivities. In addition, it
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33
34
requires providing biofunctionalization solutions allowing the grafting of several probes on the
35
36 same surface.
37
38
39
40
On the lack of standardization
41
42
43 The literature on micro and nanotechnology-based biosensors for molecular analysis is now
44
45 extremely rich. However, it is sometimes difficult to find one's way around because the
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47 experimental configurations vary so much from one transduction means to another. In an excellent
48
49
50 paper published in 2014 that reviewed the various techniques for the detection of miRNA without
51
52 amplification45, the authors presented and discussed experimental results and pros/cons of each
53
54 platform. However, there was no means to carry out a quantitative comparison of their
55
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18
1
2
3 performances (sensitivity, detection limit, analysis time, volumes required, etc.). This is largely
4
5
6 due to the lack of uniformity and standardization of the samples tested, which concerns both the
7
8 source and preparation of the samples and the choice of the biomarker. Although there have been
9
10 attempts to organize comparative tests of biosensors, such as the EILATox-Oregon Biomonitoring
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12
13 Workshop held in 20041, it seems complicated to systematize this approach and to involve the
14
15 numerous research groups active in this field. An alternative solution would be to carry out
16
17 comparative analyses using standard techniques, but even this route faces the limits of the
18
19
20 equipment currently in use (particularly in terms of limit of detection). A last path: could we
21
22 imagine having access to standard samples made available to the various research laboratories?
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24
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26
27
Conclusion
28
29 While the scientific community predicted that micro and nanotechnology would change our
30
31 approach to biology62, MEMS included, it is clear that we are still a long way from the announced
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33
34
revolution: few new medical devices today result from these technologies. Even worse, we realize
35
36 that it is impossible to propose a standard and generic technological response, as opposed to what
37
38 has been achieved by the microelectronics industry. That makes the parallel between the two
39
40
worlds complicated, even if there is a technological filiation. This feels like we have oversold the
41
42
43 impact of these technologies in the field of diagnostic aid and that we have not kept our promises
44
45 – this can be disturbing for citizens and political leaders who control the sources of funding, which
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47 is all the more detrimental at a time when the spotlight is turning on actual and future outbreaks in
48
49
50 order to answer pragmatic questions such as virological and serological tests, new treatments, new
51
52 vaccines... with the barely veiled dream of one day being able to break free from the animal
53
54 models.
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1
2
3 However, it is important to temper this analysis. Indeed, the resounding success of the in-vivo
4
5
6 glucose sensor has changed the lives of many diabetics around the world and sets an example to
7
8 follow: what an incredible advance that was just an abstract idea a few decades ago! Still, in his
9
10 excellent editorial published in August 2013 in Angewandte Chemie International Edition, Otto
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12
13
Wolfbeis wonders why the development of new molecular probes is not accompanied by the
14
15 widespread deployment of biosensors if it is only a matter of engineering, as some believe. He
16
17 notes that researchers proposing new sensors must ask themselves the right questions: “(1) Will it
18
19
be possible to monitor the evolution of the biochemical parameter over time, for example in the
20
21
22 bloodstream (...)? (2) Will the sensor operate continuously for up to 12 hours for use in surgery,
23
24 up to 2 weeks for monitoring in a bioreactor (...)? (3) Will it respond reversibly as a temperature,
25
26 oxygen or pH sensor?“63. And we should add: “Will the sensors be available within days for
27
28
29 billions of people all around the world at an affordable cost and modus operandi?”
30
31 So why is there such a gap between promises and reality? For many years, the literature trend
32
33 on new technologies biosensors was racing after the lower limit of detection, without always being
34
35
36 linked to concrete application needs and issues raised by sample composition and environment.
37
38 Fortunately, there are glimpse of hope: a good example is a HEMT (high-electron-mobility
39
40 transistor) sensor that uses a specific gate configuration to overcome the limits of electrical
41
42
43 detection, which usually requires working at low ionic strength, and which enable the measurement
44
45 of troponin I, a cardiac marker, directly in physiological solutions in a truly integrated format with
46
47 validation of the results on clinical samples using a commercial instrument64 – and we are now
48
49
50 seeing a growing number of these examples in the literature.
51
52 Thus, in order to provide tools that can assist medical decision-making as close as possible to
53
54 the patient, criteria other than sensitivity must be fulfilled: most importantly robustness and
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1
2
3 reproducibility, but also portability, low cost, minimal and generic sample preparation, and a
4
5
6 multiplex approach allowing access to precise molecular signatures with integrated reference and
7
8 calibration. In order to meet these criteria, it is important to design the tools as a whole rather than
9
10 taking care of the sole sensor and its sensitivity: how to integrate, functionalize, passivate and store
11
12
13
the sensor, how to multiplex the measurement, which sample preparation to use or adapt, how to
14
15 standardize the measurement protocols – must be the generic set of specifications for future
16
17 biosensors and, specifically, for MEMS/mechanical biosensors which so far did not demonstrate
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19
any on-field capability.
20
21
22 During the writing of this paper, among the few publications presenting biosensors based on
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24 microtechnology for the detection of SARS-CoV-2, a graphene-based field effect transistor
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26 demonstrated very promising results in terms of sensitivity65. Let’s hope these promises will soon
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29 turn into a widely commercially available device for the current COVID-19 crisis or at least to
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31 fight future pandemics. But will it withstand the test of robustness, reliability or even large-scale
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33 manufacturing in this specific case?
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40 AUTHOR INFORMATION
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43
Corresponding Author
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45 *thierry.leichle@cnrs.fr
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47
48
ORCID
49
50
51 Thierry Leïchlé: 0000-0003-3183-8976
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53 Liviu Nicu: 0000-0002-0124-7465
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55 Thomas Alava: 0000-0001-6944-0897
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3 Author Contributions
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6 The manuscript was written through contributions of all authors. All authors have given approval
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8 to the final version of the manuscript.
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13
14
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