This is the submitted version of the journal article: Pereira, Claudia; Parolo, Claudio; Idili, Andrea; [et al.]. «Paper-based biosensors for cancer diagnostics». Trends in Chemistry, Vol. 4, issue 6 (June 2022), p. 554-567. DOI 10.1016/j.trechm.2022.03.005 This version is available at https://ddd.uab.cat/record/274458 under the terms of the license 1 Paper-based biosensors for cancer diagnostics 2 Claudia Pereira,1,2,3,¥ Claudio Parolo,1,4,¥ Andrea Idili,1,5 Roger R. Gomis,6,7 Lígia 3 Rodrigues 3,8, Goreti Sales2, Arben Merkoçi1,7* 1 4 Nanobioelectronics & Biosensors Group, Institut Català de Nanociència i 5 Nanotecnologia (ICN2), Campus UAB, Barcelona, Spain. 2 6 Biomark, Sensor Research, Superior Institute of Engineering of Coimbra, Rua 7 Silvio Lima 3030-790, Coimbra, Portugal 3 8 CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 9 4710-057 Braga, Portugal 4 10 ISGlobal, Barcelona Institute for Global Health, Carrer del Rosselló 132, 08036, 11 Barcelona, Spain 5 12 Department of Chemistry, University of Rome Tor Vergata, Via della Ricerca 13 Scientifica, 00133 Rome, Italy 6 14 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of 15 Science and Technology, 08028 Barcelona, Spain. 7 16 ICREA, Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain. 17 8 LABBELS – Associate Laboratory, Braga, Guimarães, Portugal 18 *Correspondence: arben.merkoci@icn2.cat (Arben Merkoçi) 19 ¥ the authors contributed equally 20 21 Keywords 22 Paper-based sensors; Cancer screening; Early diagnosis; Point-of-care diagnostics; 23 Nanobiosensors 24 25 Abstract 26 The implementation of a wide diagnostic campaign to early diagnose cancer could save 27 millions of lives and billions of dollars every year. Unfortunately, cancer diagnostic is 28 extremely complicated and current approaches rely on the use of expensive equipment 29 and specialized personnel, which hamper their deployment in low- and middle-income 30 settings. Here, we analyze the technical challenges that must be overcome to achieve 31 precise cancer diagnostics and we describe how such hurdles have limited the 32 development of point-of-care (PoC) sensors. Then, we explain why we believe recent 1 1 achievements in the field of paper-based sensors could allow their use as widely-available 2 sensing platforms for cancer detection. Finally, we present our vision of what should be 3 done in order to make paper-based sensors widely used diagnostics platforms for cancer. 4 Global impact of cancer 5 From a global perspective, cancer is considered a disease of epidemic proportions, 6 accounting for 1 in every 6 deaths in 2018 and an estimated 9.6 million deaths, according 7 to World Health Organization (WHO) [1-3]. The introduction of new treatments has 8 allowed an improvement in the overall outcome for patients, greatly decreasing death 9 rates and increasing their quality of life [4,5]. However, this is not the real picture if we 10 observe homogeneously worldwide (Figure 1). The clinical improvement in diagnostic 11 techniques and treatments comes with higher costs and this could threat the stability of 12 healthcare systems worldwide in the next future[6]. In fact, the financial burden has 13 reached such proportions that, in some countries, physicians are forced to balance out the 14 course of testing and treatment per patient in order to minimize costs, leading to 15 ineffective diagnosis and inadequate treatments [7]. Hence, the question is: should we be 16 more focused on how to improve treatments or diagnosis? One widely accepted idea, 17 regardless the type of cancer, that has been constantly discussed, is that early diagnosis 18 holds the best chances to decrease the financial impact of cancer [8,9]. This relates with 19 the fact that patients in more advanced stages of cancer require costlier treatments, such 20 as chemotherapy or targeted therapy [10,11] 2 1 2 Figure 1: The impact of cancer on healthcare worldwide. Despite the global increase 3 in the direct costs associated to cancer, the incidence and mortality have not 4 homogenously improved, showing a great disparity between high-income and middle- 5 /low-income countries. Specifically, Asia and Africa display a lower overall cancer 6 incidence and mortality rates compared to higher-income regions such as USA and 7 Europe, and these trends are observed in both sexes. However, this pattern is the reverse 8 if we consider the Mortality to Incidence Ratio (MIR), which is found to be higher in Asia 9 and Africa compared to USA and Europe in both sexes, with men having a higher MIR 10 compared to women across all regions [12]. Besides the high cost of new treatments, this 11 disparity is also due to the need for lower-income countries to deal with other epidemics 12 related to infectious diseases, which undeniably take a good portion of the healthcare 13 budget [9,13]. Worrisomely, a projection for 2040 shows how cancer cases will increase 3 1 more in Asia and Africa than in USA and Europe. Therefore, unless this increment is 2 compensated by a MIR decrease, these areas will face an increase mortality as well. 3 4 Cancer diagnosis limitations 5 The accurate diagnosis of cancer requires the identification of three main characteristics: 6 the tumor primary site, the type of cells, and the tumor stage (Figure 2) [14]. Indeed, 7 knowing where the cancer initially developed and the type of tissue involved is essential 8 to understand how it will behave and progress [15]. Specifically, relying on the primary 9 site alone is not the most accurate strategy due to tumors’ and patients’ heterogeneity 10 (Box 1). That is, even tumor cells from the same organ can show distinct morphological 11 and phenotypic profiles that lead to a different disease progression [16,17]. For this 12 reason, pathologists routinely examine multiple sites of the same tumor in order to 13 identify the prevalent type of cells and, therefore, select the best treatment [18]. During 14 this complex process, medical doctors often rely on the use of diagnostics exams that can 15 help them to better classify the tumor. 16 Looking into diagnostics, imaging techniques and laboratory-based tests (e.g., 17 Immunohistochemistry, ELISA, PCR) use harvested tissue and blood samples to detect 18 and discriminate the cancer type [19]. Although they are still efficient and used routinely, 19 these techniques will soon become insufficient for a proper diagnosis [20]. In fact, 20 cancer’s complexity, entails the need for more accurate screening methods able to identify 21 the type and stage of cancer [20]. One example is breast cancer screening, where false- 22 positive rates in conventional mammography canbe as high as 20% [21]. Another 23 important pitfall of current techniques is their cost. For example, in the US the mean costs 24 for diagnostic mammograms are 493 dollars, for ultrasounds are 134 dollars and for 25 biopsies 2,343 dollars [22]. Additionally, considering that false negatives account for an 26 estimated 2,8 billion dollars annually, the need for more sensitive, accurate, and cost- 27 effective diagnostic tools becomes clear [22]. 4 1 2 Figure 2: Cancer classification. The TNM is the standard system for cancer malignancy 3 classification and helps understand the aggressiveness of the tumor. This system 4 considers the tumor´s morphological characteristics and translates into the classification 5 of stages from 0 to 4, where 0 is related to a tumor that has not spread, while 4 relates to 6 metastasis and high malignancy. [23]. A) The tumor’s primary site is the starting point 7 for classification. B) The system allows to assess the tumor size (T), invasion to adjacent 8 tissue/nodes (N) and possible metastasis (M) when malignant cells’ migration through 9 blood vessels to another site is implied [23]. 10 5 1 Multiplexability 2 Due to their heterogeneity, many types of cancer display abnormal levels of specific 3 biomarkers that not only are not exclusive to this disease but also present levels that may 4 vary from stage to stage [24]. For this reason, the detection of multiple cancer biomarkers 5 could help not only to determine the presence of a tumor, but also to discriminate its 6 typology and stage in a cost-effective manner [25]. Some studies have been performed to 7 validate this multiplexed approach [26,27]. For example, in 2008 a study compared the 8 accuracy of diagnosis when monitoring levels of the noncoding RNA transcript PCA3 in 9 urine alone over the monitoring of six putative prostate cancer biomarkers. [26]. The 10 authors concluded that the multiplex approach provides higher accuracy when compared 11 to the analysis of either PCA3 or prostate-specific antigen (PSA), the standard biomarker 12 for prostate cancer screening. 13 14 Sensitivity 15 If we search the literature for cancer biomarkers, we will find thousands of hits. However, 16 out of these thousands, only a small group of 20 biomarkers are FDA approved for their 17 use in diagnostic tests [28]. Indeed, finding new clinically-relevant biomarkers for cancer 18 detection is not an easy task. Looking at a molecular level, cancer status often implies 19 countless of overexpressed genes that not always correlate with over-expressed proteins 20 [29]. Moreover, since most biomarker candidates belong to pathways common to both 21 normal and oncologic tissues (e.g., angiogenesis, proliferation, and differentiation), even 22 when there is a higher expression of a given protein biomarker it may be difficult to 23 discriminate it from other physiological conditions [29]. Even when we consider a gold 24 standard technique like histology, for example, the lack of sensitivity imposes a problem 25 for early detection. For instance, although more than 15 years may pass from cancer 26 initiation to patient’s death, such time window drops to just 4 years using histology as 27 diagnostic method [29]. In fact, considering that average size of a metastatic tumor is 9 28 mm in diameter, in order to reduce mortality by 50% (e.g., in ovarian cancer) the tumor 29 should be detected when its size is only 5 mm [29]. At date, only a few expensive and 30 laboratory-based techniques (e.g. biopsies, mammography, ultrasounds) are able to detect 31 tumors of such dimensions [19]. 32 6 1 Paper-based diagnostics 2 Up until a decade ago, the required sensitivity and multiplexability for cancer diagnostic 3 were only possible by employing laboratory-based sensing platforms, such as polymerase 4 chain reaction (PCR) for genetic screening or enzyme-linked immunosorbent assay 5 (ELISA) for antigenic testing [30]. However, these technologies are not suitable for a 6 widespread use. In 2016, the WHO estimated that 1 billion people were deprived of 7 healthcare services worldwide [31]. This led several nations to reformulate public health 8 policies taking into account cost effectiveness and new sustainability goals. These 9 changes included the extensive use of PoC biosensors that adhere to the REASSURED 10 (Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, 11 User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) criteria 12 (Figure 3) [31,32]. In this context, in the last few years, paper-based devices made huge 13 steps towards bringing multiplexability and ultra-sensitivity out of the laboratory. This is 14 also reflected by their market size, with projections estimating that, for the USA alone, it 15 will reach 10.5 billion dollars by 2025 [71]. 16 7 1 Figure 3: Paper-based sensors as REASSURED devices. Although the REASSURED 2 criteria was originally formulated to define PoC sensors for infectious diseases [32] 3 (which require a prompt (ideally sub-hour) diagnosis), it can now be extended to cancer 4 diagnostics as well. During the last two decades, advances in the development of paper- 5 based diagnostic devices have allowed to achieve analytical and usability performances 6 that matches those required by the REASSURED criteria [32]. That is they have/are: 7 real-time connectivity [33], ease of specimen collection [34], affordable [35], sensitive 8 [36], specific [37], user-friendly [38], rapid and robust [39], equipment-free [40] and 9 deliverable to the end user [41,42]. Besides accuracy and usability, paper-based sensors 10 also provide great economic and environmental sustainability, supporting their use in 11 mass-scale diagnostics campaigns. 12 13 Paper-based devices for cancer biomarkers detection 14 After conducting an extensive literature research in the major peer-reviewed article 15 databases, such as PubMed, Scopus, Web of Science, and Google Scholar, we found a 16 total of 57 (Table 1 in Support Information) research studies that correspond to the search 17 in the title/abstract field of the following group of keywords: “paper-based” and 18 “biosensor” and “cancer”; “paper-based” and “sensor” and “cancer”; “biomarker” and 19 “biosensor” and “cancer”; “paper-based” and “biosensor” and “tumor”; “paper-based” 20 and “device” and “cancer”. We then excluded all the reviews and studies without 21 disclosed recognition elements or indirect cancer biomarkers. Considering that using only 22 the key words “biosensor” and “cancer” gave over 1900 hits, we consider 57 studies to 23 be a substantially low number. However, as we already mentioned, until now PoC sensors 24 have mostly been employed for the detection of infectious diseases biomarkers. Out of 25 the 57 manuscripts, only 23 tested the biosensor in human serum (Table S1, in blue), 14 26 of which are immunosensors. Moreover, none of these biosensors are considered to be 27 ready for clinical use, since they not fully adhere to the REASSURED criteria [32]. In the 28 following section, we analyze the major advances achieved to date and how they can 29 represent the foundation for a new era in cancer diagnostic. 30 31 Paper-based tests for multiplex biomarker detection 8 1 The simultaneous analysis and quantitative detection of multiple biomarkers at the point- 2 of-care is essential to achieve the required clinical sensitivity and specificity for cancer 3 diagnostics [25,27,43]. Historically, multiplexed PoC devices have suffered many 4 drawbacks, such as poor reproducibility, difficult and long optimizations, high cost and 5 potentially complicated signal read-out (especially by naked eye) [35]. However, recent 6 technological innovations in the field of paper-based sensors have overcome most of the 7 aforementioned limitations. 8 The majority of advances aiming at the development of multiplexed paper-based sensors 9 have been focused on the detection of biomarkers for infectious diseases or metabolic 10 disorders [37,44-46]. For example, Yang et al. developed a multiplexed barcoded paper- 11 based device using inkjet printing [37]. The authors claim that, by using their method, it 12 is possible to obtain barcodes with 16X higher coding ability that the standard Codabar. 13 As test bed, they detected 8 different biomarkers (for Hepatitis B Virus, Hepatitis C Virus, 14 Human Immunodeficiency Virus type 1, and human Treponema Pallidum) 8 different 15 drugs (penicillin, sulfadimidine, tetracycline, erythromycin, enrofloxacin, spectinomycin, 16 thiamphenicol, and dexamethasone) and several nucleic acid sequences (for Ebola virus, 17 hepatitis A virus Vall7 polyprotein gene, hepatitis B virus surface antigen gene, and 18 human immunodeficiency virus) in human serum samples. Using a different approach, 19 Joung et al. used a vertical assemble of functionalized paper layers to perform the rapid 20 (20 min) colorimetric multiplex analysis (13 individual immunoreactions) in patient’s 21 serum samples for the detection of Lyme-specific antibodies [45]. Furthermore, the use 22 of smartphone and cloud-based analysis provided the system with real-time connectivity 23 allowing images to be sent to a server for quick analysis and be returned back to the user. 24 On the contrary, Li et al. exploited a microfluidic 3D approach to fabricate 3D branched 25 microfluidic channels which allow to temporally resolve chemiluminescence signals. As 26 test bed for their system, they detected with good sensitivity small molecules such as 27 glucose, lactate, cholesterol, and choline [46]. Finally, Guo et al. developed a paper-based 28 fluorogenic immune-device that, by using zinc oxide nanowires (ZnO NWs), could 29 enhance fluorescence signals up to 5-folds. They demonstrated its applicability 30 simultaneously detecting 3 cardiac biomarkers (human heart-type fatty acid binding 31 protein, cardiac troponin I and myoglobin) with good sensitivity and selectivity in a 5 32 minutes test, without compromising portability [44]. 9 1 In the cancer field, we found 14 studies where the authors developed a multiplex 2 approach, 8 of which tested the biosensor in human serum. Wang et al. [47] developed a 3 chemiluminescent sensor for the detection of three cancer biomarkers (α-fetoprotein, CA 4 125, and CEA) in their clinically-relevant range. The novelty of this work relies on the 5 modification of paper with chitosan in order to achieve the covalently modification of the 6 paper surface with antibodies, therefore, conferring higher stability and robustness to the 7 device. Building up on this work, Ge et al. [48] used a fluorescent approach for the 8 detection of the same biomarkers, along with CA 153, improving the detection limits of 9 two-order of magnitude [48]. However, we must note that both works employ an ELISA- 10 like procedure (i.e., including blocking and washing steps) that compromises their use at 11 the point-of-care, being not fully user-friendly nor rapid. Another innovative work based 12 on the use of fluorescence signal is the one by Deng et al [49]. In particular, the authors 13 built a paper-fluidic chip device that allowed to detect simultaneously two small RNAs 14 sequences from tumor samples. In this work, the authors combined several innovative 15 solutions to achieve the desired analytical performance keeping a high usability. They 16 used poly(ether sulfone) to extract and purify the RNA, an hairpin probe to exponentially 17 amplify the targets, magnetic sheets to facilitate the assembly of the device and quantum 18 dots to obtain a strong fluorescence signal. 19 In the electrochemical field, Wang et al. [50] used wax- and screen-printing to fabricate 20 paper-based electrochemical aptasensors for the detection of CEA and neuron-specific 21 enolase in clinical serum [50]. Specifically, they obtained excellent analytical properties 22 (with linear ranges covering five orders of magnitude) by modifying the working 23 electrode with a combination of nanomaterials (including amino functional graphene, 24 gold nanoparticles and Prussian blue), while at the same time including features such as 25 automatic sample filtration and auto injection that facilitated the use of the device by non- 26 trained personnel. In a different work, Wang et al [51] fabricated a 3D electrochemical 27 immunosensor based on a multi-wall carbon nanotubes assemble. Using screen-printed 28 electrodes and a sandwich detection system, the HRP-O-Phenylenediamine-H2O2 allowed 29 to simultaneously detect 2 tumor biomarkers with high sensitivity [51]. Also using a 30 sandwich immunosensor, Ortega et al [52] employed a paper-based electrochemical 31 detection system to detect Claudin 7 and CD81 in extracellular vesicles. The channels 32 were fabricated using wax printing and the electrodes were printed with graphene oxide 10 1 and silver ink [52]. Remarkably, the authors successfully validated the performance of 2 their device using 80 patient samples. 3 More recently, Xu et al [53] proposed a novel sensing method for the detection of six 4 cancer biomarkers using mass spectrometry and a paper-based substrate. Specifically, 5 they developed a one-step hexaplex device that uses just 10 µL of sample for screening 6 the six biomarkers within 30 min. The paper spray ionization (PSA) was employed in 7 circle-cut papers and, along with a sandwich assemble resembling the ELISA approach, 8 the final device was able to overcome some of the challenges associated with PSA-MS 9 technique [53]. Furthermore, the authors successfully applied their method inserum 10 samples from 12 patients who have been diagnosed with cancers at several sites, including 11 breast tumors (benign and malignant), gastric cancer, and liver cancer. 12 On the bright side, we have just seen how several works successfully used paper-based 13 sensors for the multiplexing and quantitative detection of clinically-relevant biomarkers. 14 This is a crucial aspect to achieve an early cancer diagnosis as well as a personalized 15 treatment. However, there is still a margin to improve. In fact, while a few works detected 16 more than five analytes, the vast majority still detect just up to three different biomarkers, 17 which is not enough to really impact the way cancer is diagnosed. Similarly, the usability 18 of those approaches should be improved. Undoubtedly, all of them are much easier to use 19 than an ELISA, but they are probably not easy enough to be deployed at the point-of- 20 care. In this respect, we believe that including usability studies in future works will 21 strengthen the awareness that achieving excellent analytical and clinical performance is 22 not enough to have an impact in the real world. 23 24 The enhanced sensitivity of recent paper-based tests 25 The quantitative analysis of biomarkers in the sub-nanomolar range is essential to achieve 26 a precise cancer diagnostic. Looking at the past fifty years, it is clear how paper-based 27 tests have been “relegated” to the detection of highly-concentrated biomarkers. From a 28 technical perspective this is reasonable considering that most of these sensors do not have 29 incubation or washing steps. However, this limits the time window for the bioreceptors 30 to interact with the target biomarker and impeding the active removal of potential 31 interferent molecules [35]. More recently, however, advances in bioengineering and 11 1 nanotechnology have provided solutions (Figure 4) enabling to achieve remarkable low 2 detection limits and quantitative results while keeping their ease of use [36,54-59]. 3 As for multiplexing, the majority of the works achieving ultra-sensitivity in paper-based 4 devices are focused on non-cancer-related biomarkers [60]. Probably the most impactful 5 work to date is the work from Miller et al., where the authors, using spin-enhanced 6 nanodiamonds, improved the sensitivity of the biotin-avidin model in a lateral flow assay 7 over five orders of magnitude reaching the impressive limit of detection of 8.2 × 10−19 8 molar concentration [36]. Using the same set up, and following a 10-min isothermal 9 amplification step, the authors also detected a single-copy of HIV-1 RNA using clinical 10 plasma samples. The authors achieved such outstanding limits of detection by harnessing 11 the fluorescence detection of nanodiamonds. Looking at their work from a REASSURED 12 perspective, the only concern we find in the work from Miller et al. is its ease of use and 13 therefore applicability. In fact, nowadays it is possible to find commercially available, 14 miniaturized, optical sources and filters at reasonable prices [61], as well as companies 15 selling nanodiamonds of different sizes for a cost similar or lower to the one of AuNPs. 16 However, being able to detect such low target concentrations must be related to the 17 complete removal of the non-specific accumulation of nanodiamonds on the test line. 18 Currently, this is possible just by including extra washing steps that makes the whole 19 process longer and cumbersome. 20 When it comes to improving sensitivity, the use of amplification techniques is always a 21 convenient solution. In particular, due to lower cost and simplicity, isothermal 22 amplification techniques tend to be easier to integrate into point-of-care diagnostic 23 devices than the PCR. In this context, a remarkable work using the recombinase 24 polymerase amplification method is the one made by Jauset-Rubio et al., where the 25 authors developed a lateral flow assay able to detect 8.67 × 105 copies of DNA using a 26 constant temperature (37° C) in less than 15 min (including both amplification and 27 detection) [54]. Besides the analytical and usability performance, the authors designed 28 the probes in such a way that the recognition event on the paper-based test happen via 29 hybridization rather than using antibodies/hapten couples. This sensing architecture allow 30 to improve the stability of the device and its shipping and storing capabilities. Instead, 31 using the loop-mediated isothermal amplification, Suea-Ngam et al. were able to detect 32 single copies of methicillin-resistant Staphylococcus aureus (MRSA) using silver 33 nanoparticles as labels in a lateral flow assay [59]. The authors managed to integrate the 12 1 entire assay into the paper surface and used a smartphone camera for quantitatively 2 analyze the results, making the whole device portable and extremely easy to use. Finally, 3 Chen et al designed a testing platform for the detection of miR-31 using the 4 CRISPR/Cas12a system on a paper-based strip [57]. This platform combined the 5 CRISPR/Cas12a system with the “invading stacking primer” (IS-primer) amplification 6 reaction in a one-pot reaction at a constant temperature. Using their system the authors 7 could detect the target in diluted saliva without extraction procedures in 90 min at 8 attomolar levels [57]. A common criticism to the use of isothermal amplification 9 technique is the requirement of hardware that maintain a constant temperature for the 10 whole duration of the assay. Fortunately, recent advancement (such as the use of paper- 11 based batteries [62,63] allows to achieve such control at a low cost and in an integrated 12 form, making possible the use of isothermal amplification techniques also at the point-of- 13 care. 14 15 Besides optical readouts, paper-based electrochemical sensors have also achieved 16 excellent limits of detection [63] For example, Yao et al developed an ultrasensitive 17 wireless aptasensor for kanamycin detection (down to 30 fg/mL) which is based on 18 flexible freestanding graphene paper [56]. In order to achieve such low limit of detection, 19 the authors integrated in the sensor a nuclease-assisted amplification strategy, where the 20 nuclease (DNase I) allowed the catalytic recycling reaction of target. This is an important 21 aspect since, while the use of enzymes allows a dramatic amplification of the signal, when 22 employing them at the point-of-care it is always essential to verify their activity after 23 prolonged storage and shipping. In another work, , Sun et al. used molecular imprinted 24 polymers obtaining a limit of detection of 0.87 pg/mL for glycoprotein ovalbumin [55]. 25 Combining the use of nanomaterials (i.e., Au nanorods, nanoceria) and the hybridization 26 chain reaction, the authors obtained very low limit of detection However, the synthesis 27 and fabrication of several nanomaterials, as well as the use of enzymatic amplification, 28 may limit the deployment of the strategy at the point-of-care. Finally, Cinti et al. showed 29 the possibility to detect breast cancer mutations employing the signal ON and OFF 30 approach in a paper-based electrode [58]. Particularly relevant from this work is the 31 extremely ease of use of the device, requiring just the addition of the sample (in this case 32 whole blood) directly on the working electrode to obtain a quantitative readout. However, 13 1 the sensitivity of the device does not allow to achieve the detection of the biomarkers in 2 their clinical relevant range. 3 Overall, the strategies discussed to increase the sensitivity of paper-based sensors rely on 4 some external or integrated hardware which either control the temperature to achieve the 5 ideal conditions of isothermal amplifications or is required to carry out the 6 fluorescent/electrochemical measurements. However, in most cases, the hardware 7 employed has a low cost and can be easily miniaturized, making the use of this 8 amplification strategies potentially suitable for point-of-care applications, as long as they 9 maintain an adequate ease of use (as it has been the case of glucometers). In our opinion, 10 as we discussed for the work of Miller et al. [36], the main challenge will be 11 minimizing/eliminating non-specific adsorptions. However, we believe that the 12 implementation of flow control strategies such as the work of Sena et al. [34]may allow 13 to achieve ultra-sensitivity without sacrificing the ease of use. 14 1 2 Figure 4: Paper-based sensors for cancer biomarker detection. Although the use of 3 paper-based sensors for cancer detection is still at its dawn, several interesting approaches 4 have been reported. In particular efforts have been focused on addressing sensitivity and 5 multiplexing issues in order to make the results of paper-based sensors clinically relevant. 6 Regarding the sensitivity in A) Feng et al. proposed a paper-based colorimetric test that 7 supports the naked-eye detection of cancer-related miRNA down to 0.41 amol/ng in HeLa 8 cells [64]. B) In another example Moccia et al. detected down to 6 nM of cancer-related 15 1 miRNA using a paper-based electrode [65]. Regarding multiplexing C) Wu et al. have 2 reported a paper-based microfluidic electrochemical device able to carry out a radical 3 polymerization reaction that allow to achieve the detection of 4 difference cancer 4 biomarkers down to 0.01 ng/mL [66]. D) Finally, Deng et al. developed a fully-integrated 5 paper-based device capable to both extract, purify and fluorescently detect down to 3 × 6 106 copies of multiple miRNAs [49]. 7 8 Translation of the technology into clinical practice 9 The current scenario in the field of paper-based sensors is quite promising. Novel sensing 10 platforms with improved analytical performance are coming out on a monthly basis. 11 Despite this huge productivity, the development of bioanalytical devices is still mostly 12 based on a trial and error approach during the whole process of the fabrication and 13 optimization. While this approach has been fruitful so far, we should also note how the 14 improvements are generally not very significant or very rarely are successfully transferred 15 to the market. In this context, we believe that developers could take advantage of the use 16 of a more computerized approach that may allow a faster and optimized analytical 17 performance of their device [67-70]. This in turn will generate a substantial decrease of 18 the developing costs and a higher chance of getting the product to the market. Similarly, 19 the implementation of artificial intelligence models during the data analysis may result in 20 a convenient tool to improve the sensitivity and specificity of the sensor, without the need 21 to further modify the biological or hardware components [67,71]. 22 Besides the challenges inherent to technology development and the lack of a universal 23 model of diagnosis, there are socio-economic aspects to consider in order to achieve a 24 massive cancer diagnostic campaign. For example, even a high-sensitive test able to 25 detect multiple cancer biomarkers may not be able to provide a clear positive or negative 26 diagnosis per se, since variations of patient’s biomarkers may be associated to other 27 medical conditions. Therefore, the test results and other patient’s symptoms should 28 always be analyzed by a trained oncologist. In the perspective of a mass-diagnostic 29 campaign, this implies the formation and deployment of numerous healthcare 30 professionals and potentially some adaptations of current infrastructures (i.e., storage and 31 disposal of samples and tests). 16 1 Another challenge is managing the impact that such diagnosis can have in a patient’s life 2 [72]. For example, even just the idea of being tested for cancer creates such an emotional 3 stress that patients may avoid the testing altogether. At the same time, patients diagnosed 4 as positive for cancer may require psychological support that should be implemented 5 immediately after the test result. This creates the need for other new professional figures 6 that accompany the patients during the whole screening process [72]. Besides the 7 psychological burden of the testing, the patient must be guaranteed that third parties 8 cannot access the results. In fact, a breach in the data security could imply a dramatic 9 increase in the cost of health and life insurance, as well as discrimination during job 10 applications. Therefore, it seems clear that to succeed in implement a massive cancer- 11 diagnostic campaign it is essential to build around it an effective socio-economic 12 framework. 13 14 CONCLUDING REMARKS 15 Our fight against cancer cannot afford to remain stacked in current laboratory-based 16 detection methods, but must aim to implement a widespread use of point-of-care 17 diagnostic devices that adhere to the REASSURED criteria. In this respect, paper-based 18 sensors appear to be the most viable solution since they combine excellent analytical 19 performance with economic and environmental affordability. Specifically, we showed 20 how recent advances in the field of nanotechnology allowed the development of 21 multiplexed and ultra-sensitive paper-based devices that have the potential to provide the 22 required clinical sensitivity and specificity for cancer diagnostic. Accompanied with the 23 right ethical and care management, this could decrease the economic burden of healthcare 24 systems and improve the outcomes of cancer patients. 25 26 27 28 29 BOX 1 – Diagnostics for cancer personalized medicine 17 1 In addition to the difficulty to early identify the type and stage of cancer, the idea that one 2 treatment fits all does not take into consideration the biological uniqueness of each 3 patient. In chemotherapy, for example, it is crucial to control the drug dosage in order to 4 maximize its effectiveness and minimize secondary effects[73]. Indeed, the metabolic 5 and physiological differences of individuals affect differently the pharmacokinetics of 6 drugs and they are often not considered in the posology [73]. This leads to unnecessary 7 side effects due to the under- and over-dosing of the therapeutic drugs, which increase 8 direct, informal and indirect costs [7]. To overcome this, medical care has been driven 9 towards personalized medicine with the idea to customize the standard procedures to a 10 patient’s specific needs [74]. Nowadays, roughly half of the oncological drugs have an 11 associated diagnostic kit to assure the drug is administrated only to patients that can 12 benefit from it (i.e., using genetic tests)[75]. As healthcare switches from fee-for-service 13 (a fee per service rendered) to value-based (payment based on patients’ health outcome) 14 medicine, proper analytics will be key to improve treatments’ success that is translated 15 into a more cost-effective health system [76,77]. 16 In cancer, current personalized medicine relies tremendously on genetic and antigenic 17 testing [78,79]. The former provides high levels of specificity and sensitivity; however, 18 it requires long experimental protocols, the need of specialized personnel, and expensive 19 equipment which hampers its use on a wide scale. Contrarily, the latter can potentially be 20 used in point-of-care (PoC) sensors providing rapid and quantitative results within 21 minutes, and without the need of specialized personnel; however, their low accuracy and 22 sensitivity has often prevented their use for the detection of biomarkers in the low and 23 sub-picomolar range (concentrations often required to achieve early diagnosis) [80]. It 24 appears clear that a technology capable to provide enough sensitivity and specificity at 25 the point-of-care could revolutionize the way how clinicians can perform cancer 26 diagnosis. However, to achieve such accuracy, it is paramount to find the right 27 combination of biomarkers in order to provide the desired specificity and a sensing 28 technology able to quantify their concentrations in their clinically-relevant ranges. 29 30 31 32 ACKNOWLEDGMENTS 18 1 This study was supported by the Portuguese Foundation for Science and Technology 2 (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and 3 BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European 4 Regional Development Fund under the scope of Norte2020 - Programa Operacional 5 Regional do Norte. Claudia Pereira was recipient of a fellowship supported by a doctoral 6 advanced training (call NORTE-69-2015-15) funded by the European Social Fund under 7 the scope of Norte2020 - Programa Operacional Regional do Norte. 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In blue are highlighted the studies conducted in real samples. Recognitio Type of Signal detection n element Biomarker LOD / Range Approach Ref paper nature 0.1 - 35.0 ngmL- Covalently immobilize Chemi- α-fetoprotein 1 antibodies on µPADs; Chitosan luminescence Antibodies CA 125 0.5 - 80.0 UmL-1 [47] Sandwich CL-ELISA on ELISA CEA 0.1 - 70.0 ngmL- 1 µPADs; 5x10-4- 100 ngmL-1 Two polyethylene α-fetoprotein 1x10-4- 100 terephthalate substrates, with CA 125 UmL-1 one paper layer with the Chitosan Amperimetric Antibodies [81] CA 153 1x10-4- 100 sensing sites and the other CEA UmL-1 layer with counter electrode 5x10-4- 100 and reference electrode. ngmL-1 Microfluidic paper-based Whatman 0.001 – 75.0 CA125 wax-patterned device based chromatograp Electrochemical Antibodies UmL-1 [51] CEA on multi-walled carbon in a hy paper 1# 0.05- 50 ngmL-1 sandwich mode nanotubes 0.001 - 100 ngmL-1 Covalently immobilization α-fetoprotein 0.0002 - 100 Whatman of capture antibodies; CuO CA 125 UmL-1 chromatograp fluorescence Antibodies NPs-labeled secondary [48] CA 153 0.0002 - 100 hy paper 1# antibody in a sandwich-type CEA UmL-1 immunoreaction; 0.005 - 200 ngmL-1 0.001 -100 ngmL-1 α-fetoprotein 0.001 -100 SiO2 nanoparticles in which Whatman CA 125 ngmL-1 antibody and HRP were co- chromatograp Electrochemical Antibodies [82] CA 153 0.005 -100 immobilized were used as the hy paper #1 CEA ngmL-1 signal reporter 0.005 -100 ngmL-1 0.01 -100 ngmL- 1 α-fetoprotein 0.05 -100 ngmL- Use of graphene to modify Whatman 1 CA 125 the immunodevice surface to chromatograp Electrochemical Antibodies [66] CA 153 0.05 -100 ngmL- accelerate the electron hy paper 1# 1 CEA transfer; 0.01 -100 ngmL- 1 Sequential growing gold -1 nanoparticles and manganese Whatman 0.0012 ngmL ; oxide nanowires networks on chromatograp Electrochemical Antibodies PSA 0.005 - 100 [83] a freestanding three hy paper 1# ngmL-1 dimensional origami flexible device; Au-paper electrode Whatman HL-60 cells 4 cells/10 µL; fabricated and employed as chromatograp Electrochemical Aptamers Anti-cancer 500 – 7.5x107 the working electrode; [84] hy drugs cellsmL-1 Sequential in-electrode 3D paper #114 cell culture 26 Pre-loads capture antibodies Whatman Electrochemical Antibodies PSA 6 pgmL-1 onto a paper disk serving as a [85] nitrocellulose small electrochemical cell. Two hydrophilic cells patterned connected by a Electro- 1.0pgmL-1; carbon ink, with enhanced Not disclosed chemiluminescen Antibodies PSA 1.0pgmL-1 - 100 response through [86] ce ngmL-1 modification of the bipolar electrode cathode with multi- walled carbon nanotubes Microfluidic paper-based Whatman Electro- MCF-7 cells 250 cellsmL-1; electro-chemiluminescence chromatograp chemiluminescen Aptamers 450 - 107 by origami construction with [87] -1 hypaper#114 ce cellsmL aptamers functionalized with 3D macroporous Au-PCE 500 cellsmL-1; Whatman nanocompos 7.5x102 – Gold nanorods modified ITO Impedance K562 cells [88] filter paper #1 ite 3.9x106 cellsmL- electrodes 1 Composite of poly(3,4- 25.8 μAng- Whatman 1 -2 ethylenedioxythiophene):pol Electrochemical Antibody CEA mLcm ; 2-8 [89] filter paper #1 y(styrenesulfonate) and ngmL-1 reduced graphene oxide Nitrocellulose Graphene oxide-gold paper nanoparticles-anti-EpCAM Infrared (Whatman Antibody MCF-7 cell 600 cells; composite added into [90] temperature gun AE99 and mid nitrocelulose with CLW-040) immobilized target cells Lanthanide-doped 0,89 ngmL-1; upconversion nanoparticles CEA Filter paper Fluorescence Antibody 0 – 100 ngmL-1 tagged with specific [91] antibodies printed to the test on the test paper Device with microfluidic channel built through wax Whatman 10 ng mL-1; printing and electrodes chromatograp Electrochemical Antibody CEA 50 pgmL-1 - 500 through screenprinting; [92] hy paper 1# ngmL-1 Working electrodes modified with NH2-G/Thi/AuNPs nanocomposites Isothermal amplification through a netlike rolling circle amplification 9.3 fM; pH strips Colorimetric NA miR-21 technique; quantification [64] 20 fM - 20 nM through correlation between hydrogen ions production and miR-21 concentration Extraction and purification of Whatman small RNA through 3x105 copies chromatograp miR-21 polyethersulfone paper chip; Fluorescence DNA probes 3x105 to 3×108 [49] hy paper, miR-155 downstream detection was copies grade 2 performed with Quantum dots as signal labels. NH2-G/Thi/AuNPs-modified Electrochemical Neuron- Whatman #1 10 pg mL-1; μPADs, combined with Smartphone Antibody specific [93] filter paper 1 - 500 ng mL-1 electrochemical detector and readout enolase Android's smartphone, 27 Boronic acid modified N, Glucosamine Boronic acid and carbonyl S co-doped 1.3 µM at 340 Filter paper Naked eye in cancer cell groups structurally rigid N-S [94] carbon nm HepG2 doped carbon quantum dots quantum dots Immobilized aptamer linked quantum dots and Cy3 250 pM labeled complementary Not disclosed Fluorescence Aptamer EpCAM [95] 1 – 100 nM DNA Carbon ink-based closed bipolar electrode with wax printed reaction zone and use Whatman #1 Electro- 40 cellsmL-1; of AuPd nanoparticles chromatograp chemiluminescen Aptamer MCF-7 cells 100 - 106 [96] -1 serving as both as a capture hy paper ce cellsmL aptamer carrier and as catalyst for the reaction of luminol and H2O2. Paper working electrodes 0.005 -100 Whatman modified with gold Antibodies CEA ngmL-1 chromatograp Electrochemical nanoparticles and [97] peptide PSA 0.001 – 40 hy paper #1 cyclodextrin functionalized ngmL-1 gold nanoparticles A rotatable paper- photocontrollable switch that photoelectrochem CEA in 0.001 - 200 ng allows light source to Not disclosed DNA probe [98] ical MCF-7 cells mL-1 selectively activate desired working zones of the cyto- sensor AuNPs functionalized with Filter Positive/Negativ specific antibody conjugated Naked eye Antibody p16INK4a [99] Nitrocellulose e result with HRP for increased signal Use of ionic liquid to minimize unspecific binding and secondary antibodies 0.51 ngmL-1 Whatman stacked on the surface of the Colorimetric Antibody CEA 0.002 - 75.0 [100] paper carboxylated CO2(OH)2CO3- ngmL-1 CeO2 nanocomposite with intrinsic peroxidase-like activity DNA hybridization coupled BRCA-1 0.4 µM for a 250 Nitrocellulose Fluorescence DNA probe with a smartphone based [101] gene dilution factor readout system Gold nanoparticles labelled Whatman with biotinylated ssDNA 3 pgmL-1 chromatograp Colorimetric Antibody PSA sequences and streptavidin- [102] hy paper HRP for enzymatic signal enhancement. Polyhedral AuPd alloy 20 cellsmL-1 Electrochemical nanoparticles and three- Not disclosed Aptamer MCF-7 cells 50 - 106 cellsmL- [103] and colorimetric 1 dimentional reduced graphene oxide Sheet-type biosensor based 1.18 ngmL-1 Micro-pore on a multi wall carbon Electrochemical Antibody PSA 0.002 - 75.0 [104] filter paper nanotubes activated with a ngmL-1 PSA antibody 28 Erbium ion (Er3+)-doped and thulium ion (Tm3+)-doped PSA 89 pgmL-1 upconversion nanoparticles Not disclosed Colorimetric Antibodies [105] EphA2 400 pgmL-1 as two independent reporters on two-color lateral flow strips Gold nanoparticles/reduced Whatman 10 pgmL-1; graphene oxide/thionine chromatograp Electrochemical Aptamer PSA 0.05 - 200 nano composites to coat [106] hypaper#1 ngmL-1; working electrodes for aptamer immobilization Cysteine-capped gold Whatman Modified 0.4 µM; nanoclusters on Y shaped Colorimetric Citrate [107] filter #1 aminoacid 1.0 μM -10 mM paper-based microfluidic device 10.2 μAng- Whatman 1 paper modified with Electrochemical Antibody CEA mLcm-2 [108] filter paper #1 nFe2O3@PEDOT:PSS/WP 4 - 25 ngmL-1 Biosensor composed by a miR-21 first layer of silver nanowire Filter paper Electrochemical Aptamer 0.1 fM [109] miR-141 and a second layer MoS2/AuNPs. Inactivated uriase printed onto paper microzones. DNA HF180 TP53 gene containing C−C mismatches nitrocellulose Naked eye Enzyme 11 nM [110] missmatch allows reactivation of urease membrane activity, hydrolysis of urea and alteration of pH Nanocomposites of reduced graphene 0.01 UmL-1 Pure cellulose Electrochemical Antibody CA125 oxide/thionine/gold [111] 0.1 - 200 UmL-1 nanoparticles coated onto working electrodes Gold nanorattles coated with cetyltrimethylammonium - urine chloride functionalized with Filter paper Plasmonic Antibody 50 to 5x103 [112] perilipin2 monoclonal antibody by pgmL-1 means of a bifunctional polyethylene glycol Composites of graphene- thionin-AuNPs and of Prussian-blue-PEDOT- CEA 0.01-500 ngmL-1 Not disclosed Electrochemical Aptamers AuNPs were synthesized in [50] NSE 0.05-500 ngmL-1 order to promote electron transfer and the targets’ aptamers immobilization A silver 0.78 U/mL nanoparticles/reduced Not disclosed Electrochemical Antibody CA-125 [113] 0.78 – 400 U/mL graphene oxide nano-ink on flexible paper substrate Presence or Signal ON and OFF in a filter absence of paper-based waxed strip that Filter paper Electrochemical DNA probe H1047R [58] missense uses gold nanoparticles to mutation anchor the recognition probe Paper modified with Whatman 1.06 ngmL-1 graphene and poly (3,4- Electrochemical Aptamer CEA [114] filter paper #3 0.77–14 ngmL-1 ethylenedioxythiophene):pol y(styrenesulfonate) 29 A customized PNA probe on a paper surface with a two Office paper 6 nM layer wax print and a Electrochemical Peptide miRNA-492 [65] sheet 10 - 100 nM nanostructured working electrode surface modified with gold nanoparticles. (SERS)-based biosensor 1.79 pgmL-1 composed of gold Cytochrome Filter paper SERS Aptamer 0 pgmL-1 –10 nanourchins, an aptamer and [115] c µgmL-1 a complementary DNA labelled with Cy5 Chip manufactured with Nitrocellulose NMP22 Positive or paper and wax; Dual-channel Glass cellulose Fluorescence Antibody [116] BTA negative µPAD with colloidal gold Filter paper immune labeling Microfluidic paper-based 3.07 × 1015 M laser-induced sensor that miRNA 21 3.28 × 1015 M Filter paper Fluorescent Aptamers uses duplex-specific [117] miRNA 31 in A549 cell nuclease for fluorescent lysates signal amplification A sponge-like polyvinyl alcohol pad and Calcine- Chromatograp 0.34 fgmL-1 preloaded dry filter paper Colorimetric Primers PCA3 [118] hic paper were placed in the amplification and detection zones of a 3D-printed chip 1.054 ngmL-1 α-fetoprotein 0.1 – 100 ngmL- AuNPs conjugated with 1 Nitrocellulose Colorimetric Antibodies cysteamine immobilized on a [119] MUC 16 0.413 ngmL-1 nitrocellulose membrane 0.1 – 10 ngmL-1 Biotinylated aptamer for OPN pre-capture, immobilized antibody on the 0.1 ngmL-1 test line for second specific Nitrocellulose Colorimetric Aptamer Osteopontin 10 - 500 ngmL-1 target identification and [120] membrane streptavidin-modified gold nanoparticles on the conjugation pad for color detection. Use of trans cleavage activity of CRISPR/Cas13a to mediate subsequent Electro CRISPR exponential amplification Not disclosed chemiluminescen miRNA 17 10-15 M [121] RNA and a “light-switch” ce molecule [Ru(phen)2dppz]2+ to avoid electrode modification and washes Standard graphene-gold nanoparticle Fluorescence Antibodies uPA 100 pM [122] copier paper platform with quantum dots 1 ng mL-1 Use of the properties of gold Not disclosed Colorimetric Antibodies PEAK1 10-9 - 10 -6 gmL- nanoparticle in color dye [123] 1 degradation A DNA-templated Ag/Pt Whatman Colorimetric DNA probe miRNA-21 10 – 103 pM nanoclusters with peroxidase [124] filter #1 mimetic activity. 30 Surface of working Chromatograp 10 pg mL-1 electrodes modified amino- hy Paper Electrochemical Antibody VEGF-C 0.01 – 100 functional single-walled [125] (Whatman #1) ngmL-1 carbon nanotubes and gold nanoparticles CA 15-3 25 UmL-1 Gold nanoparticles CA 19-9 40 mUmL-1 functionalized with Mass CEA 5 ngmL-1 antibodies for a imuuno Not disclosed Antibodies [53] spectrometry CA 125 35 UmL-1 sandwich assay with readout HE4 140 pmolmL-1 through online mass α-fetoprotein 10 ngmL-1 spectrometry 0.4 pgmL-1 A dual sandwich-type Claudin 7 2 – 103 pgmL-1 Whatman immunosensor that employ Electrochemical Antibodies 3 pgmL-1 [52] paper #1 reverse phase proteomic CD81 0.01 – 10 ngmL- 1 array 1 31