This is the submitted version of the journal article: Rubio-Monterde, Ana; Quesada-González, Daniel; Merkoçi, Arben. «Toward integrated molecular lateral flow diagnostic tests using advanced micro- and Nnanotechnology». Analytical chemistry, Vol. 95, issue 1 (october 2023), p. 468-489. DOI 10.1021/acs.analchem.2c04529 This version is available at https://ddd.uab.cat/record/271938 under the terms of the license Towards integrated molecular lateral flow diagnostic tests using ad- vanced micro- and nanotechnology Ana Rubio-Monterde1,2, Daniel Quesada-González1*†, Arben Merkoçi1,2,3,4* 1 Paperdrop Diagnostics S.L., MRB, Campus UAB, 08193 Bellaterra, Spain 2 Nanobioelectronics and Biosensors Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, Barce- lona, Spain 3 The Barcelona Institute of Science and Technology (BIST), Campus UAB, 08036 Bellaterra, Barcelona, Spain 4 ICREA, Institució Catalana de Recerca i Estudis Avançats, Pg. Lluis Companys 23, 08010, Barcelona, Spain CONTENTS INTRODUCTION Introduction Since the COVID-19 pandemic, some of the routine meth- In vitro diagnostic medical devices ods for clinical diagnosis of infectious diseases have become closer to the general population and terms like “PCR” (pol- Lessons learnt from COVID-19 pandemic ymerase chain reaction) and “antigen rapid diagnostic test Non-covered medical needs and new biomarkers (RDT)” are not restricted only to the scientific community Lateral Flow biosensors but now also belong to the lay language. Given the emer- From immunochromatography to rapid diagnostic gency, the pandemic also raised awareness about supply tests politics and regulation of the market for diagnostic tests (e.g., Emergency Use Authorization, EUA, from the Food and Working principle Drug Administration, FDA; or the "Conformité Européenne" Types of LF tests according to bioreceptor used marking, CE marking, from the European Union, EU). PCR Tests in the market and RDT are two major techniques currently used in the clinical practice for diagnosis of diseases. On the one hand, Nucleic Acid Amplification Techniques PCR is a highly sensitive method, but relies on expensive Polymerase chain reaction (PCR) equipment and qualified personnel. On the other hand, Technique rapid diagnostic tests, despite being less sensitive, are Types faster, cheaper, portable, and battery/instrument-free. In this review, we will discuss the advantages and disad- Tests in the market vantages of rapid antigen tests and different nucleic acid Isothermal era amplification techniques (NAAT), such as PCR, both as in What is isothermal amplification? vitro diagnostic devices. To do so, our review explains the Loop-mediated isothermal amplification working principle of each method, classifies the different (LAMP) subtypes and analyzes the impact that will have the intro- duction of isothermal amplification methods in the diagnos- Rolling Circle Amplification (RCA) tic field. In addition, this review gives an overview of the Nucleic Acid Sequence Based Amplifica current available products in the market and the new trends tion (NASBA) generated as a result of combining the strengths of NAAT Recombinase Polymerase Amplification and RDT. (RPA) In vitro diagnostic medical devices Strand Displacement Amplification (SDA) From May 26th, 2022, when the “Regulation (EU) 2017/746” came into force1,2 replacing Directive Nicking Enzyme Amplification Reaction 98/79/EC, the development and use of diagnostic tests in (NEAR) the EU are controlled by the new regulatory framework. Ac- Tests in the market cording to this regulation, in vitro diagnostic medical de- New trends vices are considered any medical device which is a reagent Conclusions (or a reagent product), control material, kit, instrument, cal- ibrator, apparatus, piece of equipment, system or even a software, being used in solitary or combined, which its in- tended use (according to the manufacturer) is for an in vitro application. The regulation also implies that the device is intended for the examination of specimens, (which includes transmissible stage.13 RDT were questioned for their sensi- human tissue and blood donations), solely or mainly for tivity15,16 but allowed fast detection of infected patients, at- providing information of relevance of at least one (or more) home testing,17 and high-frequency testing due to its low of the following cases: (i) a physiological or pathological cost, which overall helps to prevent spread to others. Figure process or state; (ii) congenital physical or mental impair- 114 shows a schematic of the optimal timeframe to use the ments; (iii) the predisposition to a medical condition or a different types of tests and the viral dynamics of SARS-CoV- disease; (iv) the determination of the safety and compatibil- 2 infection in a symptomatic patient. Equally important to ity with potential recipients; (v) the prediction of the treat- acknowledge advantages and disadvantages of every test18, ment response or reactions; (vi) the definition or monitor- is to understand the test performance and its intended use, ing therapeutic measures.1 because no test is 100% accurate.10 This definition includes from specimen receptacles to ma- Non-covered medical needs and new biomarkers laria, viral loads, or prenatal tests, and the now so well- known COVID-19 tests. Some of these tests are based on The ongoing SARS-CoV-2 pandemic has highlighted the PCR or rapid immunochromatography, which are tech- importance of being able to develop, produce and deploy niques that have been used in clinical practice for decades3– rapidly diagnostic tests for emerging infectious diseases.19 6, but the COVID-19 pandemic made vox populi. SARS-CoV-2 will not be the last threat, as other viruses like avian influenza (Influenza H5)20 or monkeypox21 have Lessons learnt from COVID-19 pandemic proven recently. However, not only systems for detection In December 2019, an outbreak caused by “Severe acute and surveillance of viruses are needed, the increasing prev- respiratory syndrome coronavirus 2” (SARS-CoV-2) was alence of antimicrobial resistance (AMR) has become a si- identified in Wuhan,7,8 soon after, the virus rapidly spread lent pandemic which has been exacerbated by the SARS- and became a global pandemic declared by the World CoV-2 pandemic.22 AMR is a serious burden and threat to Health Organization (WHO) on March 11th. In a short time, global public health,23–25 according to the Centers for Dis- the need for available and reliable diagnostic tests was un- ease Control and Prevention (CDC), only in the United der an increasing demand and a flood of laboratory-devel- States, every year more than 2.8 million antibiotic-resistant oped tests (LDTs)9,10, EUA tests10 and CE marked tests infections happen, and because of them more than 35,000 reached hospitals and laboratories. By end of July 2020, the people die.24 Particularly concerning is the spread of multi- FDA had authorized 163 COVID-19 diagnostic tests under resistant bacteria, also known as “superbugs”.25 The WHO EUA.10 Those diagnostic methods for detection of SARS- has recognized the urgency for new in vitro diagnostics tests CoV-2 were classified in (i) molecular tests, such as the PCR, to combat AMR. Among the tests required are: (i) RDT to and (ii) antigen tests, also known as RDT.11,12 differentiate between bacterial and viral infections; (ii) tests to identify pathogens; and (iii) antimicrobial suscepti- PCR of nasopharyngeal samples became the gold stand- bility tests (AST).26 ard test for the diagnosis of COVID-1911, but despite its lower limit of detection (LOD) it usually implies transport Another gap in the market that needs to be addressed is of the sample to a central laboratory to be analyzed by qual- the rapid diagnosis of sepsis. Sepsis is a life-threating con- ified staff, high cost and, consequently, low-frequency test- dition responsible of 1 in 5 deaths worldwide,27 defined ing, which entails a delay of some days to obtain the re- as an organ dysfunction caused by a dysregulated host re- sult13,14 and has de risk of being positive after the sponse to infection.28,29 The lack of a reliable and fast diag- nostic tests plays a part in the use of broad-spectrum Figure 1: Timelines for optimal use of different diagnostic tests for Covid-19 detection and host response. Reprinted with permis- sion from Peeling, R. W.; Heymann, D. L.; Teo, Y. Y.; Garcia, P. J. Diagnostics for COVID-19: Moving from Pandemic Response to Control. Lancet 2022, 399 (10326), 757–768, (ref. 14). Copyright ® (2022) Elsevier. antimicrobials, which contributes to increase the preva- LATERAL FLOW lence of AMR and this at the same time complicates the sep- LF biosensors are RDT based on paper. These tests are sis treatment.29,30 Blood culture remains as the gold stand- low-cost, portable, easy-to-use, and equipment/battery- ard for sepsis diagnostic, however it is time-consuming free, characteristics which make them highly suitable for (which can delay critical therapeutic decisions) and has POCT, low-resource settings and emergency use. poor sensitivity. Culture-independent techniques (e.g. mo- lecular methods and mass spectrometry) may overcome From immunochromatography to rapid diagnostic tests some of the these limitations,30,31 but they are not tech- Paper chromatography, invented by Martin and Synge in niques suitable for point-of-care testing (POCT) or low-re- 1943, can be considered the first paper-based sensor.46 source settings. The WHO wish list for the properties of the Later, in 1970s, the first LF test was commercialized: the sepsis diagnostic test needed includes: (i) rapid identifica- pregnancy test, which is one of the most used POCT.47,48 Fol- tion of pathogen type (viruses, bacteria, fungi, parasites); lowing this milestone, the first infectious disease LF test (ii) high sensitivity and specificity to guide antimicrobial was commercialized in the late 1980s for the detection of treatment and limit antimicrobial overuse; (iii) no need to Group A Streptococcus pyogenes from throat swabs sam- culture (iv) detection of multiple pathogens at the same ples.49 From there, LF biosensors extended widely to other time; (v) detection of AMR (vi) easy to use; and (vii) low fields including veterinary, environmental control, food price.29 safety, diagnosis and others.50 Apart from the lack of diagnostic tests, the difficulty to di- In the last decade, the inclusion of nanomaterials lead to agnose sepsis relies in the absence of a validated bi- better performance with lower LOD and other benefits.51–54 omarker.28,31 There is special interest in biomarkers that Nanomaterials (those of 100 nm size or smaller ) have prop- could be detected in the emergency room (ER) or critical erties that diverge from those of the same materials at the care POCT to provide rapid results, and therefore rapid and macroscopic scale, presenting phenomena such as electro- adequate treatment.28 Non-coding RNAs, particularly mi- magnetic field enhancement, quantum confinement, and croRNAs (miRNAs), and extracellular vesicles (EVs) are ex- plasmonic resonance, which can be exploited for signal am- amples of potential biomarkers that are being investigated plification.53 There are recent reviews devoted to nano- to aid in sepsis diagnostic.28,31,32 Outside the market of infec- materials and their promising applications in LF, which are tious diseases, miRNAs and EVs are also emerging as a dis- too wide to be included in this review. Refer to the reviews ease biomarkers, standing out in cancer diagnose and liquid of Nguyen et al.,55 Sena-Torralba et al.,52 Vahid Shirshahi and biopsy.33,34 EVs are lipid-bilayer membrane structures se- Guozhen Liu,56 and Mirica et al.57 for more information creted by cells which have an important role in cell-to-cell about novel nanomaterials employed in LF tests. It must be communication via the transfer of nucleic acids (NAs), pro- highlighted that gold nanoparticles (AuNPs; a.k.a. colloidal teins and lipids that can affect physiological processes.34 gold) are the most used nanomaterial on LF tests due its Among the NAs transferred there are miRNAs, which are high biocompatibility (easy to conjugate with bioreceptors), small non-coding RNAs, 21-25 nucleotides long, that have easiness and reproducibility in synthesis, tuneability (size, an important role in gene expression regulation. miRNAs shape and color) and strong colorimetric response. Other are emerging as non-invasive biomarkers for a variety of nanomaterials as carbon nanoparticles and upconverting diseases such as cancer, endometriosis or cardiovascular nanoparticles (e.g. Eu-based nanoparticles),39 also present- and infectious diseases.33–38 The current methods for ana- ing fluorescence capabilities, are becoming popular in re- lyzing miRNAs are reverse transcription qPCR (RT-qPCR), search papers but not in the same way (yet) in commercial Norther blot-based systems, microarrays, in situ hybridiza- systems, as later shown in Table 1. Large scale production tion and next-generation sequencing (NGS). Each of these for novel nanomaterials and their use in commercial test techniques have their advantages and disadvantages but all could still be challenging. of them are far from being POCT platforms. This unmet need could be covered by isothermal amplification assays Recently, the global outbreak of SARS-CoV-2 has been a and RDT based on Lateral Flow (LF), which hold potential boost for the use of RDT based on LF, which has improved as POCT systems for miRNA detection.38–40 the trust in these tests in the diagnostic field.50 The LF tests success is also reflected in the commercial market, consid- POCT is defined as testing performed at or near the site of ering that the global market for LF tests was valued at USD patient care with rapid turnaround time of test results to 8.2 billion in 2020 and is projected to reach USD 11.7 billion the patient41. POCT are developed to fulfill the ASSURED by 2028, growing at a CAGR (compound annual growth (Affordable, Sensitive, Specific, User-friendly, Rapid & ro- rate) of 4.6% from 2021 to 2028.58 bust, Equipment-free, and results Delivered to the end-us- ers) guidelines of the WHO.42 As proven during the SARS- Working principle CoV-2 pandemic, POCT can improve healthcare in different Briefly, the physical principle of the LF assays is based on aspects, from enabling earlier disease detection and moni- capillarity action. When the extracted specimen is added to toring to reaching low-resources settings.43,44 From cardiac the sample pad, the flow goes through the paper reaching markers to infectious disease testing, the menu of POCT is the conjugate pad, where the nanoparticles (NPs) conju- wide and growing, by 2025 the global POCT market is ex- gated57 with the bioreceptor are stored, ready to react to the pected to exceed US $44.6 billion with a 9% of compound target analyte (if present). Once the flow reaches the detec- annual growth rate.44 RDT and molecular POCT systems are tion pad (membrane), if the specimen contains the target, candidates to fulfill this demand in the near future.45 the test line (TL) will appear indicating a positive result due to the presence of a specific bioreceptor that recognizes the target. Therefore, if there is no target on the sample, the TL will not appear visible. The sample continues flowing aptamers63–65 can be integrated depending on the target to be detected. In LF immunoassays (LFIA), monoclonal and polyclonal antibodies are the more common bioreceptors applied. Nevertheless, recently nanobodies and antibody fragments are standing out due to some advantages such less unspe- cific binging and low batch-to-batch variability in compari- son with monoclonal and polyclonal Ab.52,59 The usual target of LFIA are of protein nature66 (e.g., hormones, antigen virus or bacteria, toxins), although other targets such as drugs of abuse67, cofactor molecules68 or even heavy metals69 can be also detected. However, despite its high applicability in LF, Ab present some limitations including high batch-to-batch variability, long development time, need for ethical approval and re- frigerated storage.63,70 In addition, there are some non-im- munogenic targets like small molecules (e.g. antibiotics, bacterial spores, pesticides) for which is difficult to obtain high-affinity Ab. Aptamers, also called “chemical Ab”, have been investigated as bioreceptors for LF to address these Figure 2: Components and working principal of a LF test. a) The restrictions from the Ab.70 main parts of a LF strip are the sample pad, the conjugate pad, Aptamers are single-stranded DNA (ssDNA) or single- the membrane (where the bioreceptors are printed) and the ab- stranded RNA (ssRNA) molecules that fold in a complex 2D sorbent pad, all of which are assembled on a laminated card. b) or 3D structure and bind to a target with high affinity and and c) working principle of a classical LF immunoassay b) The selectivity.71,72 They have several clear advantages over Ab, presence of the target in the sample leads to the appearance of including high stability, in vitro generation, ease in function- two red lines due to the accumulation of nanoparticles on TL and alization, and capacity of large scale synthesis.63,73 Aptamers CL. c) In the absence of the target, only the CL appears. Reprinted are developed by an in vitro iterative process called “sys- with permission from Parolo, C.; Sena-Torralba, A.; Bergua, J. F.; tematic evolution of ligands by exponential enrichment” Calucho, E.; Fuentes-Chust, C.; Hu, L.; Rivas, L.; Álvarez-Diduk, R.; (SELEX),73 which has been modified depending on the tar- Nguyen, E. P.; Cinti, S.; Quesada-González, D.; Merkoçi, A. Tuto- rial: Design and Fabrication of Nanoparticle-Based Lateral-Flow get of interest74,75,76. By SELEX or its variants, aptamers have Immunoassays. Nat. Protoc. 2020, 15 (12), 3788–3816 (ref. 59). been generated for hormones, drugs, heavy metals, pro- Copyright © (2020) The Authors (Quesada-González, D. and teins, toxins, enzymes, cancer cells, and bacterial, viral and Merkoçi, A.), under exclusive license to Springer Nature Limited. fungal pathogens.73 Aptamer-based LF tests use aptamers as bioreceptors, yet they do not solve the lack of sensitivity through the detection pad and a control line (CL), contain- of the LF70 and their binding activity is highly dependent on ing a secondary bioreceptor, should appear for all valid the ionic strength of the buffer and the presence of certain tests. Finally, an absorbent pad is located at the end of the cations.59 strip for absorbing the fluid excess (Figure 2).59 Non structured nucleic acids (i.e., DNA probes) are also The same principle applies in the multiplexed LF assays, used as bioreceptors. There are Nucleic Acid Lateral Flow which allow simultaneous detection of more than one tar- (NALF) tests, also called DNA-based LF tests62, and Nucleic get. The most straightforward way to develop a multiplexed Acid Lateral Flow Immunoassay (NALFIA).49,77 In NALF, dif- LF is just by printing multiple TL with the corresponding bi- ferent DNA probes are conjugated to the NPs and printed in oreceptor in the detection pad. However, the number of tar- TL and CL. These DNA probes are designed to be comple- gets to be multiplexed by this mechanism is limited by the mentary according to Watson-Crick base pairing to the tar- length of the detection pad and/or the different labels avail- get (e.g., a miRNA or genetic amplicon, which is the product able. Recent advances and novel strategies have been devel- of a molecular amplification, with primers extended with a oped lately to address these challenges (e.g. microarrays, ssDNA tail). In the case of NALFIA, dual labeled targets (pro- fluidic devices or the use of different NPs in the same as- teins and genetic amplicons) typically with biotin and an- say).52 other antigen with high Ab affinity, i.e., digoxigenin or fluo- Types of LF tests according to the bioreceptors used rescein, are captured in a sandwich complex between biotin and streptavidin in one side, and antigen and antibody im- The bioreceptors are biological compounds used for the munoreactions in the other.78 capture of the target and, in the case of LF tests, for its recog- Test in the market nition by means of a sandwich format (Figure 2). There is a minimum of three bioreceptors needed for this design of a Apart from the huge distribution of LFIA (antigen rapid LF test: (i) one labelled with the NPs; (ii) another one tests) for SARS-CoV-279 detection (709 CE mark by October printed in the detection pad as a TL; and (iii) the last one 2022),80 LF tests play a role in the diagnosis of other pathol- that is able to capture the first bioreceptor or other mole- ogies and as POCT or in the ER. The following table 1 sum- cules at the surface of the NPs and is printed as a CL.59 marizes the portfolio of some of the most relevant compa- The classic bioreceptors used in LF tests are antibodies nies with in vitro diagnostic LFIA products available nowa- (Ab), but other bioreceptors such DNA probes40,60–62 or days. In the table it can be observed that colloidal gold, AuNPs, are the most common nanomaterial in LF tests, as we previously said, together with latex beads (often above the 100 nm, thus not being considered nanomaterials). Latex beads need to be dyed in order to produce color and their surface must be functionalized for their conjugation (often with carboxylic groups), while AuNPs can directly interact with thiolated bioreceptors, among others. It must not go unnoticed that those kits using fluorescent nano- or microparticles (it includes Eu beads) will always require the use of an additional equipment for reading the results, while using other reporters the response, in a qualitative manner, could be read by naked-eye (however, the equipment may help to register the data or even quantify the concentration of analyte). Table 1. Portfolio of relevant companies with in vitro diagnostic LFIA and its application field. Company Brand name Reporter Reader Application field of the as- Regulatory approval says SD BIOSENSOR STANDARD F Europium beads STANDARD F 2400 Respiratory Depending on the assay RUO STANDARD F 200 Serology and/or CE STANDARD F 100 Vector borne Gastrointestinal Hepatitis Blood borne Chronic disease Inflammation Cardiovascular Hormones Tumor marker SD BIOSENSOR STANDARD Q Colloidal gold No Respiratory Depending on the assay CE Serology Vector borne Blood borne Hepatitis Gastrointestinal Cardiovascular Abbot BinaxNow™ Colloidal gold DIGIVAL™ Respiratory antigens Depending on the assay CE, Urine FDA and/or EUA Malaria G6PD deficiency Abbot PANBIO™ Colloidal gold No Respiratory antigens Depending on the assay CE Dengue and/or FDA Serology Company Brand name Reporter Reader Application field of the as- Regulatory approval says Operon Rapid tests Colored latex particles OPERON Lateral Flow Respiratory All CE Reader Gastrointestinal Food intolerance Tumor markers Pregnancy Hepatitis CerTest Biotec Rapid test Colored latex particles No Fecal antigens All CE Respiratory antigens Urine Inflammatory and tumor markers R-Biopharm AG RIDA®QUICK Colored latex particles or RIDA®QUICK SCAN II Enteropathogenic infections All CE colloidal gold NG-BIOTECH NG-Test® Colloidal gold NG-Test® Reader Antibiotic resistance Depending on the assay CE Respiratory and/or FDA Pregnancy Serology CORIS BioConcept RESIST (for the antibiotic re- Colored latex particles or No Respiratory All CE sistance product line) colloidal gold Enteric Gastric Antibiotic resistance Drug Serology QUIDEL Sofia Fluorescent microparticles Sofia 2 Respiratory Depending on the assay CE, Gastrointestinal FDA, EUA and/or CLIA waived Lyme Pregnancy BD Veritor™ Colloidal gold Veritor System Respiratory Depending on the assay CE, FDA, EUA and/or CLIA waived RUO: Research use only. FDA: Approved by FDA by premarket notification 510(k), De Novo or Premarket Approval Application (PMA). EUA: Emergency use authorization only by FDA. CE: European conformity, can be CE-IVDD by the Directive 98/79/EC on in vitro Diagnostic Medical Devices or CE-IVDR by the European conformity by Regulation (EU) 2017/746 (IVDR) on in vitro diagnostic medical devices. CLIA waived: Clinical Laboratory Improvement Amendments waiver, which means classified as “simple laboratory examinations and procedures that have an insignificant risk of an erroneous result” by FDA according to requirements (42 CFR Part 493). uses a thermostable polymerase, which allows the enzyme NUCLEIC ACID AMPLIFICATION TECHNIQUES to withstand the heating required for the reaction and main- Nucleic acid amplification technologies (NAATs) are tain its activity.81–83 based on sequence-specific recognition and amplification of PCR reaction follows three main steps repeated in cycles: a region of interest in the genome of targets to be detected. denaturation, annealing, and extension (Figure 3a).84 Dena- The most common NAAT used in in vitro diagnostic is the turation takes place at 94-96 ºC and it separates the double- PCR, which is a laboratory technique developed by Kary stranded DNA (dsDNA) template into ssDNA. Next, the tem- Mullis in 1983 that can make millions of copies of a specific perature is lowered between 45 and 60 ºC, allowing the pri- DNA region in a short time. PCR presents some advantages mers to hybridize to their complementary site on the DNA and drawbacks in comparison to RDT, despite being highly template (annealing). The union of the primers provides a sensitive, it relies on qualified personnel and expensive place for the polymerase to attach and begin copying the equipment. However, during the last decade, other NAAT template, usually at 72 ºC (extension). These steps are re- based on isothermal amplification (IA) have gained atten- peated many times in cycle using an automated thermal cy- tion, especially for POC applications. cler resulting in the exponential amplification of the DNA Polymerase chain reaction (PCR) between the primers. ,82,83 In cases where the sample is RNA, the PCR is preceded by the conversion of the RNA to a com- Technique plementary DNA (cDNA) by a reverse transcriptase (RT- PCR is a powerful technique used to produce multiple PCR), and then de cDNA enters the PCR cycles (Figure 3c).84 copies of a nucleic acid region of interest.4,81 The use of more than one primer pair allows to amplify The four main components of a PCR are the DNA tem- multiple DNA regions simultaneously in one PCR reaction, plate, the primers, the nucleotides (deoxynucleotide tri- resulting in a multiplex PCR.4 phosphates, dNTPs), and a thermostable DNA polymerase: Types (i) The DNA template is the sample which contains the re- gion of DNA to be amplified; (ii) primers are short oligonu- When the results of the PCR are analyzed at the end of the cleotides DNA sequences, custom designed to be comple- reaction it is called end-point PCR (or end-point RT- mentary to the target sequence; (iii) dNTPs are the material PCR85,86). Typically, this is performed using an agarose gel to synthesize new DNA strands, they are single units of Ad- electrophoresis, where the samples are ran together with a enine (A), Thymine (T), Cytosine (C) and Guanine (G); (iv), ladder to check if the amplicon size (i.e. the number of base the DNA polymerase is an enzyme which synthesizes new pair, bp) obtained is the one expected.82 With the corre- DNA strands complementary to the target sequence, PCR sponding primer modification, the PCR result can also be read by a NALF o NALFIA.78,87,88 Figure 3: Schematic representation of the a) PCR process, b) TaqMan Probe Real Time PCR and c) RT-PCR. Modified with permission from Dunbar, S.; Das, S. Amplification Chemistries in Clinical Virology. J. Clin. Virol. 2019, 115, 18–31 (ref. 84). Copyright © (2019) Elsevier. On the other hand, Real Time PCR (qPCR or RT-qPCR) (sample-to-result). This trend is also reaching dPCR with enables detection and measurement of products generated the first cartridge plus automated systems receiving regula- as the amplification progresses. There are several chemis- tory approvals. Some of these platforms aim at single or few tries used for the detection of PCR product during real-time pathogens detection (multiplex) while others follow a syn- PCR89, being one of the most applied the use of TaqMan® dromic approach (simultaneously target multiple patho- probes, which are designed to hybridize within the target gens with overlapping symptoms), offering panels of sev- sequence. These probes are labelled by a fluorophore and a eral pathogens being simultaneously tested. quencher, once they hybridize, they are cleaved by the DNA Isothermal era polymerase during the extension resulting in the separation of the quencher from the fluorophore, which start to emit What is isothermal amplification? fluorescence that is monitored by an instrument.4,89,90 The inclusion of a standard curve in the qPCR allows Recent advanced methods in NAATs are based on isother- quantification of the target by extrapolating the amplifica- mal amplification (IA), which overcomes the need of ther- tion curve during the exponential phase of the reaction it- mal cycling given the ability to amplify the sequence of in- self.89,91,92 However, digital PCR (dPCR) is a method for ab- terest at constant temperatures.95,96 By eliminating the need solute quantification of the DNA/RNA of interest based on of thermocyclers, amplification techniques are set free from end-point fluorescence measurement without standard energy-demanding and technologically complex systems, curve.92,93 In dPCR, first the sample is separated into many opening the door to POCT and in-field applications.96–100 IA independent PCR sub-reactions and, depending on the techniques are thus suitable for laboratories lacking expen- method used for this partition, dPCR is classified as droplet sive PCR equipment and of low-resource settings. In addi- based dPCR or chip-based dPCR.4,93,94 Following the split, tion, IA methods require less sample and reagent prepara- the PCR takes place and finally, the fluorescence signals are tion, and the results can be read through different ap- detected and quantified based on Poisson distribution. Alt- proaches (e.g., turbidimetry, LF), enhancing their accessibil- hough dPCR has many advantages when compared to qPCR, ity and ease-of-use.98,99,101 such as higher reproducibility, accuracy and tolerance to in- IA methods are becoming more and more popular, in a hibitors, it still has major disadvantages as the high cost and way that in the near future we could be entering the isother- complex instrumentation.93,94 mal era as IA defies PCR. IA techniques differ from one an- Figure 4 summarizes the main differences, already dis- other in features such as the enzymes used, number of pri- cussed, between end-point PCR, qPCR and dPCR.92 mers, time and temperature of amplification or the template required. Nevertheless, the future of IA methods is still de- Tests in the market pendent on fulfilling technical maturity and broader com- mercialization of enzymes and reagents, which will come PCR is widely distributed in the in vitro diagnostic market with the expiry of the corresponding patents.99,102–106 in different setups (e.g., closed systems, automated robots, syndromic testing, etc.). Table 2 compiles some of these sys- As the field expands, there are several IA already pa- tems available today for in vitro diagnosis (IVD). tented, like the Crossing Priming Isothermal Amplification (CPA)102 and the Helicase Dependent Amplification Table 2 does not include IVD PCR assays to be use with (HDA),107 or under development as the Signal-Mediated common thermocyclers such as Bio-Rad Real Time PCR de- Amplification of RNA Technology (SMART).98,99 These last tection systems, the LightCycler or the StepOne from Ap- IA methods will not be described within this review since plied Biosystems among others. However, form its infor- the available information or the predicted impact is still lim- mation we can observe how many PCR platforms consist of ited. Instead, we briefly describe the most noteworthily and a cartridge and automated instruments, resulting in walka- currently having most potential to be part of in vitro diag- way solutions where the user only needs to add the sample nostic tests. Figure 4: A comparison of end-point PCR, qPCR and dPCR. Reprinted with permission from Quan, P. L.; Sauzade, M.; Brouzes, E. DPCR: A Technology Review. Sensors 2018, Vol. 18, Page 1271, 2018, 18 (4) (ref. 92). Copyright © (2018) MDPI. Table 2. Some of the remarkable PCR platforms for IVD commercially available. Platform Company PCR Detection Setup Singleplex/Multi- Assays Regulatory name technology technology plex/Syndromic approval ePlex GenMark PCR Electrochemical Sample-to-result Syndromic Respiratory Pathogen Panel 1 CE and FDA RT-PCR Cartridge and instru- Respiratory Pathogen Panel 2 CE and EUA ment BCID-GP Panel CE and FDA BCID-GN Panel CE and FDA BCID-FP Panel CE and FDA ARIES® Luminex RT-qPCR Real Time fluo- Sample-to-result Multiplex SARS-CoV-2 EUA and CE qPCR rescence Cartridge and instru- Bordetella CE and FDA ment C. difficile CE and FDA Flu A/B & RSV CE and FDA Group A Strep CE and FDA Group B Strep CE and FDA HSV 1&2 CE and FDA MRSA FDA Norovirus CE STANDARD SD BIOSENSOR RT-qPCR Real Time fluo- Sample-to-result Multiplex MPX/OPX RUO M10 qPCR rescence Cartridge and instru- MDR-TB CE LAMP ment MTB/NTM CE HPV CE C. difficile CE Arbovirus panel CE Flu/RSV/SARS-CoV-2 CE SARS-CoV-2 CE SARS-CoV-2 turbo CE GeneXpert Cepheid RT-qPCR Real Time fluo- Sample-to-result Multiplex Xpress CoV-2/Flu/RSV plus CE and EUA qPCR rescence Cartridge and instru- Xpress CoV-2 plus CE and EUA ment Xpress SARS-CoV-2/Flu/RSV CE and EUA Xpress SARS-CoV-2 CE and EUA Xpress Strep A CE, FDA and CLIA waived Xpress Flu/RSV CE, FDA and CLIA waived Xpress Flu FDA and CLIA waived MRSA NxG CE and FDA SA Nasal Complete CE and FDA Platform Company PCR Detection Setup Singleplex/Multi- Assays Regulatory name technology technology plex/Syndromic approval MRSA/SA BC CE and FDA MRSA/SA SSTI CE and FDA Carba-R CE and FDA Norovirus CE and FDA EV CE and FDA C. difficile BT CE C. difficile/Epi FDA vanA/vanB CE vanA for VRE FDA MTB/RIF CE and FDA MTB/RIF Ultra CE MTB/XDR CE Ebola CE and EUA CT/NG CE and FDA HPV CE GBS CE and FDA Trichomonas vaginalis CE and FDA Resistance Plus® MG FleXible CE HBV Viral Load CE HCV Viral Load CE HCV VL Fingerstick CE HIV-1 Qual XC CE HIV-1 Viral Load XC CE Bladder Cancer Detection CE Bladder Cancer Monitor CE Breast Cancer STRAT4 CE BCR-ABL Ultra CE and FDA FII & FV CE and FDA BCR-ABL Ultra p190 CE FilmArray bioMérieux PCR Fluorescence Sample-to-result Syndromic Respiratory Panel 2.1 CE and FDA RT-PCR (melting curve) Cartridge and instru- Respiratory Panel 2.1 plus CE ment Respiratory Panel EZ FDA and CLIA waived Respiratory Panel 2.1 EZ EUA Blood Culture Identification CE and FDA Platform Company PCR Detection Setup Singleplex/Multi- Assays Regulatory name technology technology plex/Syndromic approval Gastrointestinal Panel CE and FDA Meningitis/Encephalitis Panel CE and FDA Pneumonia Panel CE and FDA Pneumonia Panel Plus CE Joint Infection Panel CE and FDA QIAstat-Dx Qiagen RT-qPCR Real Time fluo- Sample-to-result Syndromic Respiratory Panel CE and FDA rescence Cartridge and instru- Respiratory SARS-CoV-2 CE and EUA ment Viral Vesicular Panel RUO Gastrointestinal Panel CE SARS-CoV-2/FluA/B/RSV CE Meningitis/Encephalitis Panel CE Savanna® Quidel RT-qPCR Real Time Fluo- Sample-to-result Syndromic Respiratory Viral Panel-4 CE rescence Cartridge and instru- ment Accula™ Mesa Biotech RT-PCR LF Sample-to-result Singleplex or Multi- FluA/FluB CE, FDA and CLIA waived (now Ther- (Oscar™) Automated and self- plex RSV CE, FDA and CLIA waived moFisher) contained system with Strep A FDA and CLIA waived cassettes and dock sta- SARS-CoV-2 EUA tion cobas® Roche RT-qPCR Real Time Fluo- Assay tubes and auto- Multiplex Influenza A/B & RSV CE and FDA Liat® qPCR rescence mated instrument Strep A CE and FDA SARS-CoV-2 EUA Influenza A/B & SARS-CoV-2 EUA C. difficile CE and FDA cobas® Roche RT-qPCR Real Time Fluo- Sample-to-result Multiplex Babesia CE and FDA 6800/8800 qPCR rescence Huge, fully integrated, BKV CE and FDA and highly auto- CHIKV/DENV CE mated instruments for CMV CE and FDA high throughput CT/NG CE and FDA Flexible workflow and the ability to test multi- DPX CE ple assays in each run EBV CE and FDA HBV CE and FDA HCV CE and FDA HEV CE HIV-1 CE and FDA Platform Company PCR Detection Setup Singleplex/Multi- Assays Regulatory name technology technology plex/Syndromic approval HIV-1/HIV-2 Qualitative CE and FDA HPV CE and FDA Influenza A/B & RSV UC CE MAI CE MPX CE and FDA MTB CE MTB-RIF/INH CE SARS-CoV-2 CE and EUA SARS-CoV-2 and Influenza A/B CE and EUA SARS-CoV-2 variant RUO TV/MG CE and FDA WNV CE and FDA Zika CE and FDA QXDx AutoDG Bio-Rad RT-ddPCR Fluorescence Automated Droplet Multiplex BCR-ABL CE and FDA ddPCR Generator system con- SARS-CoV-2 EUA sisting of QXDx Droplet Reader and QXDx Au- tomated Droplet Gen- erator Previous NA extraction and thermocycler needed Dr. PCR™ Optolane Tech- Digital RT- Fluorescence Cartridge + LOAA ana- Multiplex BCR-ABL1 CE nologies PCR lyzer FastPlex Tri- PreciGenome RT-ddPCR Taqman fluores- DropX-2000 digital Multiplex SARS-CoV-2 EUA plex SARS-CoV- cent probe PCR system and the as- 2 Detection Kit sociated microfluidic cartridge Previous NA extraction RUO: Research use only. FDA: Approved by FDA by premarket notification 510(k), De Novo or Premarket Approval Application (PMA). EUA: Emergency use authorization only by FDA. CE: European conformity, can be CE-IVDD by the Directive 98/79/EC on in vitro Diagnostic Medical Devices or CE-IVDR by the European conformity by Regulation (EU) 2017/746 (IVDR) on in vitro diagnostic medical devices. CLIA waived: Clinical Laboratory Improvement Amendments waiver, which means classified as “simple laboratory examinations and procedures that have an insignificant risk of an erroneous result” by FDA according to requirements (42 CFR Part 493). Loop-mediated isothermal amplification (LAMP) Features like the possibility of being performed in a heat- ing block and interpreting the results by naked eye make LAMP was first published in 2000,108 after being pa- LAMP suitable as a POCT.116 In addition, it is reliable for low tented,105 and since then it has been successfully applied to resource clinical settings that lack minimal expertise and detection of a variety of pathogens, food control and cancer equipment. However, the complex primer design, the related mutations.99,100,109 One of its most relevant acknowl- chance to obtain false positives (due to hybridization by edgements has been its approval by the WHO as an alterna- primers) and the difficulty to multiplex (due to the number tive molecular diagnostics test for pulmonary tuberculosis of primers required) still need to be addressed before LAMP (TB).99,110 is routinely applied as an in vitro diagnostic test.97 LAMP consists of a single amplification period from 60 to Rolling Circle Amplification (RCA) 65 ºC with a turnaround time (TAT) of less than 1 hour. LAMP assays require 4 or 6 primers (these include inner RCA is inspired on the rolling circle replication that hap- and outer primers), that recognize 6-8 different regions of pens in plasmids and viruses in vivo, is an enzyme-based IA the target. To do so, it makes use of a strand-displacing DNA that can be performed from room temperature to 60 ºC with polymerase from Bacillus stearothermophilus (Bst), which a TAT of maximum 90 min.99 RCA needs a strand-displace- initiates the DNA synthesis (Figure 5a).98,111 The results of ment DNA polymerase, a primer (RNA or DNA), a target (cir- the amplification can be interpreted by naked eye visual- cular template) and dNTPs. RCA employs enzymes such as izing the turbidity of the medium97 or by other methods Bst, Phi29, and Vent exo-DNA polymerases, but Phi29 DNA such agarose gel, electrochemistry, real-time fluorescence polymerase is the most used.98 Briefly, RCA is performed by or LF strips.112–115 a strand-displacement DNA polymerase when a specific pri- mer binds to a circular template in presence of dNTPs and Figure 5: Working principle of some Isothermal Amplification techniques a) LAMP, adapted with permission from Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res. 2000, 28 (12), e63–e63. (ref. 111). Copyright © (2000) Oxford University Press. b) (I) RCA, (II) HRCA, (III) PG-RCA, (IV) multiply primed RCA, (V) C2CA, modified with permission from Yue, S.; Li, Y.; Qiao, Z.; Song, W.; Bi, S. Rolling Circle Replication for Biosensing, Bioimaging, and Biomedicine. Trends Biotechnol. 2021, 39 (11), 1160–1172. (ref. 120). Copyright © (2021) Elsevier. c) NASBA, adapted with permission from Zanoli, L. M.; Spoto, G. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices. Biosens. 2013, Vol. 3, Pages 18-43 2012, 3 (1), 18–43. (ref. 122). Copyright © (2013) MDPI. generates a long ssDNA or ssRNA molecule with tandem re- Recombinase Polymerase Amplification (RPA) peats (Figure 5b).98,99,117 In order to be able to amplify a lin- ear template, padlock probes were introduced. Padlock RPA requires three different enzymes (recombinase, sin- probes118 are linear oligonucleotides that become circular- gle stranded binding protein (SSB), and DNA polymerase) ized when the ends are joined together by ligase enzyme, and two primers.97,98 Depending on the detection method, which happens when they hybridize with a specific se- exo, nfo and fpg probes can be used.98 Adding more sets of quence. RCA results can be interpreted using agarose gel primers, RPA offers the feasibility of multiplex detection of electrophoresis, visually using fluorescence spectroscopy, more than one target in a single reaction.97 The reaction microscopy and flow cytometry97,98 or with LF strips. starts with the recombinase, which binds to single stranded 114,119,120 nucleic acids, the primers, in the presence of ATP, and tar- gets the complementary double strand sequence in the tem- RCA method results in a linear amplification of the target. plate. When the primer hybridizes with the template, a D- Several modifications of RCA have been developed: hyper- loop structure is formed that allows a strand displacement. branched rolling circle amplification (HRCA), primer gener- The displaced DNA strand is then coated by SSB while pri- ation-rolling circle amplification (PG-RCA), multiply primed mer extension by DNA polymerase forms a new dsDNA RCA and circle-to-circle amplification (C2CA),99,120 (Figure which acts as a template for the next cycle of amplification 5b) allowing exponential amplification. (Figure 6a).98,99,126 The primers are longer than the standard The low temperature profile of RCA makes it interesting PCR primers, between 30-36 bp,95,97 the amplicon size is for POCT and it has been used for biosensing applications recommended to be less than 500 bp, the temperature for such detection of bacteria or miRNA99,119,120,121,117, however, the whole protocol is 37-42 ºC, and amplification time from no RCA based diagnostic test have yet reached the market. 10 to 20 minutes with a TAT of 20 to 40 min. Gel electropho- Nucleic Acid Sequence Based Amplification (NASBA) resis, fluorescence methods for real-time and LF strips can be used to detect the amplified product.61,62,98,127 NASBA is an isothermal transcription-based technique, RPA has been applied for detection of human, animal and that mimics the retroviral RNA replication, with a TAT of 1.5 plant pathogens, including virus, bacteria and parasites, in to 2 hours. The method requires two primers (P1 and P2) singleplex and multiplex assays.98,99 The ease of the proce- and three thermolabile enzymes (T7 RNA polymerase, dure, the low working temperature and the short time re- RNase H, and reverse transcriptase). NASBA is more suita- quired for amplification makes RPA a suitable candidate ble for RNA targets, but it can be applied to DNA targets add- technique for integration into in vitro diagnostic devices ing two denaturation steps at 95 °C. through microfluidics as POCT or in-field testing.98,126–128 NASBA mechanism involves two phases: (i) Initiation The main drawback of this method is that the RPA rea- phase (non-cycling), where the target RNA is converted to gents are only sold by one company, TwistDxTM Limited (Ab- dsDNA by reverse transcription; and (ii) amplification bot), who owns the patent.103,129 Other limitations are that it phase (cycling), where the dsDNA molecules are actively is prone to produce unspecific signals due to primer-dimer transcribed into RNA products (Figure 5c).99 The standard formation and that real time RPA is dependent on RPA ki- NASBA protocol starts with an incubation step (for RNA at netics.129 65 ºC and for DNA at 95 ºC) to denature the target and then the following amplification takes place at 41 ºC. In the initi- Strand Displacement Amplification (SDA) ation phase, the forward primer (P1), which includes the This IA technique needs an exonuclease-deficient DNA promoter sequence for T7 RNA polymerase, hybridizes to polymerase (typically Bst DNA polymerase or Klenow frag- any RNA target present in the sample and is extended by the ment), a restriction endonuclease HincII and four different reverse transcriptase. Next, the RNA strand of the resulting primers (one pair, E1 and E2, with single-stranded re- RNA:DNA hybrid is destroyed by RNase H, and the reverse striction site overhangs, and a second pair known as primer (P2) hybridizes to the complementary sequence fol- “bumper primers” to support the displacement of the am- lowing production of dsDNA by the reverse transcriptase, plification product from the first set of primers).95,98 The obtaining a dsDNA with the target sequence and a T7 pro- protocol starts with the annealing of a primer in a DNA moter. Then, the T7 RNA polymerase produces multiple breathing site of the template95,130 or by denaturing the RNA copies that are complementary to the target RNA. In dsDNA (~95 ºC) to allow hybridization with the template of the amplification phase, each newly synthesized RNA can be E1 and E2, which have recognition sites for the restriction copied, resulting in an exponential amplification of RNA enzyme and are extended to produce a strand containing complimentary to the target.98,122 those sites. These strands are next displaced by the bumper Once amplification is complete, the amplified products primers, resulting in dsDNA and displaced single strands can be detected by several ways, such as: molecular bea- from E1 and E2 extensions (Figure 6b). The new nicking se- cons, fluorescence markers, gel electrophoresis, enzyme- quences are recognized and cleaved by HincII, generating a linked gel assay, electro-chemiluminescence, LF or electro- nick with an extendable 3′ end. The strand-displacing poly- chemical detection.98,99,114,122 merase extends from the nick, causing the displacement of NASBA assays have been developed for detection of a di- the single-stranded template sequence, where the primers versity of virus, bacteria, and parasites.99,123–125 However, will hybridize again.95 The exponential amplification is ac- the method is limited by the need of thermolabile enzymes complished by coupling sense and antisense reactions, in after the denaturation step, and the difference between the such a way that the displaced sense strand serve as tem- initial temperature for denaturation (65 °C for RNA and 95 plate for the antisense reaction and vice versa.99,131 The TAT °C for DNA) and the temperature for the amplification reac- is around 2.5 h and the results are interpreted through gel tion (41 °C).98 electrophoresis, fluorescence methods, molecular bea- cons98,99 or LF.114,130 The low temperature required for the isothermal amplifi- fragments in minutes (Figure 6c). The nicking, polymeriza- cation (between 30 and 55 ºC) makes it prone to unspecific tion, and strand displacement is repeated at 60 ºC resulting products due to unspecific primer-binding,99 in line with the in linear amplification,95 but there is also an exponential other IA methods presented. Nevertheless, SDA has been version of the method (EXPAR), where the product also acts successfully applied for the diagnosis of tuberculosis (TB), as a primer.99 The products generated are ssDNA and can be Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) detected using molecular beacons or other probes based and herpes simplex virus (HSV) from urogenital sam- methods and LF.84,135 ples.98,99 One of the limitations of the technique is the capa- The main limitation of the NEAR technique is the for- bility to amplify sequences only between 50 bp to 120 bp mation of a large number of non-specific products, which long,95 but on the other hand this feature makes it suitable inhibit the progression of the forward reaction and compro- for miRNA profiling. 98,99,132 mise the detection sensitivity, so it therefore may need Nicking Enzyme Amplification Reaction (NEAR) more specific amplification analysis methods.84,133,135 Other shortcoming is the primer design, which needs to include a NEAR,106 also called nicking enzyme assisted amplifica- restriction site, a stabilizing region, and a target-binding re- tion (NEAA) or nicking enzyme mediated amplification gion that is complementary to the template.133,135 (NEMA),133 combines the use of a strand-displacing DNA Tests in the market polymerase and a nicking endonuclease. The endonuclease creates a nick that enables the polymerase to initiate ampli- This increasing interest in the IA techniques is also being fication, rapidly producing many short nucleic acids from reflected in the market, as some in vitro diagnostic tests the target sequence.134 This process is extremely fast and based on IA are reaching commercialization. Table 3 pre- sensitive, allowing detection of small DNA or RNA sents these new acquisitions of the market. Figure 6: Working principle of some Isothermal Amplification techniques. a) RPA, adapted with permission from Li, J.; Macdonald, J.; Von Stetten, F. Review: A Comprehensive Summary of a Decade Development of the Recombinase Polymerase Amplification. Analyst 2019, 144 (1), 31–67. (ref. 126). Copyright © (2019) Royal Society of Chemistry. b) SDA, modified with permission from Toley, B. J.; Covelli, I.; Belousov, Y.; Ramachandran, S.; Kline, E.; Scarr, N.; Vermeulen, N.; Mahoney, W.; Lutz, B. R.; Yager, P. Isothermal Strand Displacement Amplification (ISDA): A Rapid and Sensitive Method of Nucleic Acid Amplification for Point-of-Care Diagnosis. Analyst 2015, 140 (115), 7540–7549. (ref. 130). Copyright © (2015) Royal Society of Chemistry. c) NEAR, adapted with permission from Wang, L.; Qian, C.; Wu, H.; Qian, W.; Wang, R.; Wu, J. Technical Aspects of Nicking Enzyme Assisted Amplification. Analyst 2018, 143 (6), 1444–1453. (ref. 133). Copyright © (2018) Royal Society of Chemistry. Table 3. Main in vitro diagnostic systems based on IA. Platform name Company IA technology Detection Setup TAT Assays Regulatory technology approval PortNAT® Ustar Biotechnolo- CAP LF all-in-one test cassette 40 min COVID-19 CE gies VerosTM Sense Biodetection Isothermal expo- LF all-in-one test cassette 15 min COVID-19 CE nential amplifica- tion ID NOWTM Abbott NEAR Real time fluo- Cartridges + instrument Max. 13 min FluA/FluB CE and EUA rescence RSV CLIA waived Strep A 2 CLIA waived COVID-19 CE and EUA Solana Quidel HDA Fluorescence Pre heat sample lysis 25 to 60 min de- Bordetella CE and FDA Cartridges + instrument pending on the as- C. difficile CE and FDA say Group A Strep CE and FDA Group B Strep CE and FDA HSV 1+2/VZV CE and FDA Influenza A+B CE and FDA RSV + hMPV CE and FDA SARS-CoV-2 CE and EUA Trichomonas CE and FDA NUCLISENS® bioMérieux Real time NASBA Beacon probes Big instrument (8 to 48 sam- 60 to 150 min de- HIV CE and FDA EASYQ® ples at the same time) pending on the HPV CE sample number Enterovirus CE and FDA MRSA CE and FDA eazyplex® Amplex Diagnostics LAMP Real time fluo- Sample pretreatment 15 to 30 min de- SARS-CoV-2 All CE GmbH rescence Freeze dried ready to use pending on the as- Candida auris amplification reaction to be say BloodScreen used with instrument STDs GENIE® II Pneumocystis jirovecii PneumoBug expert CSF direct SuperBug CRE SuperBug complete SuperBug expert SuperBug Acineto Platform name Company IA technology Detection Setup TAT Assays Regulatory technology approval SuperBug AmpC SuperBug basic MRSA VRE EHEC TyphiTyper C. difficile ProbeTec™ ET BD Diagnostics SDA Fluorescence Big instrument Up to 46 patient CT/GC All CE and FDA System samples in 3 hours Chlamydia trachomatis Neisseria gonorrhoeae CT/GC Trichomonas vaginalis HSV Alethia® Meridian Bioscience LAMP Turbidity Sample heat pretreatment Less than 1 hour HSV type 1 & 2 CE and FDA Meridian Alethia Incubator / C. difficile CE and FDA Reader CMV CE and FDA Group A Strep CE and FDA Group B Strep CE and FDA Mycoplasma pneumonia CE and FDA Bordetella pertussis CE and FDA Malaria CE Chlamydia trachomatis CE Neisseria gonorrhoeae CE LoopampTM Eiken Chemical LAMP Visual fluores- Strip tubes with lyophilized 40 min Malaria CE cence reagents Mycobacterium tuberculosis CE SARS-CoV-2 CE mfloDx™ MDR- EMPE Diagnostics RCA LF Amplification kit + LF kit 75 min approx. Mycobacterium tuberculosis Under clinical TB complex and mutations in evaluation the rpoB, katG and inhA genes FDA: Approved by FDA by premarket notification 510(k), De Novo or Premarket Approval Application (PMA). EUA: Emergency use authorization only by FDA. CE: European conformity, can be CE-IVDD by the Directive 98/79/EC on in vitro Diagnostic Medical Devices or CE-IVDR by the European conformity by Regulation (EU) 2017/746 (IVDR) on in vitro diagnostic medical devices. CLIA waived: Clinical Laboratory Improvement Amendments waiver, which means classified as “simple laboratory examinations and procedures that have an insignificant risk of an erroneous result” by FDA according to requirements (42 CFR Part 493). As can be seen in table 3, LAMP is, for the moment, the IA for assays being developed by other companies or research method most used in the commercialized tests. Other wor- centres. thy observation is that half of these platforms are still in- Coupling an IA technique with a LF platform as a readout strument-dependant, mostly because the detection technol- allows developing POCT with the benefits of paper-based ogy chosen is by fluorescence. biosensors and the sensitivity of the molecular assays. Pre- sent and future research on these molecular LF tests will fo- NEW TRENDS cus on the integration of the whole testing process into a Recently, molecular assays and LF tests have intersected microfluidic system,77,144–146 novel nanomaterials as labels at the diagnostic field, particularly for POCT, with LF being (a.k.a. transducers or reporters)39,52 and amendment of the used as the readout technology for nucleic acid testing.49 specificity of the IA methods. The Accula™ system from Mesa Biotech (now Ther- As previously said, AuNPs clearly stand out as labels for moFisher),136,137 the PortNAT® system from Ustar Biotech- LF tests due their easiness to be integrated in the assays. nologies138,139 and the VerosTM test from Sense Biodetec- However, fluorescent nanomaterials may help reaching tion140 prove it, as they are the first integrated molecular LF lower LOD and higher sensitivities, which would permit to in vitro diagnostic tests in the market. The three of them use quantify the results with improved resolution, but on ex- NAAT, integrated in a cassette, and the results are display in change of the dependence of an external reader. Carbon na- a LF strip with a simple workflow of one/two steps from the noparticles, as the ones employed in Veros™ (Table 3), have sample, being suitable as a POCT. demonstrated improved performance compared with The Accula™ system has assays for FluA/FluB, respira- AuNPs,52,55 and their synthetic routes may be more environ- tory syncytial virus (RSV), Group A Streptococcus (strep A) mental friendly and sustainable, however the conjugation and SARS-CoV-2 detection. Accula™ Flu A/Flu B, RSV and with bioreceptors is more complicated and the scalability is Strep A tests have received 510(k) clearance and Clinical still very challenging. Laboratory Improvements Amendments (CLIA) waivers Exciting developments have been made in the integration from the FDA. Flu A/Flu B and RSV tests have also obtained of LF and IA technologies147 by means of microfluidics. To CE Mark in the EU. Additionally, the Accula™ SARS-CoV-2 quote some examples, E. A. Phillips et al.148 developed a mi- test has obtained EUA from the FDA. Accula™ system uses a crofluidic rapid and autonomous analysis device (micro- PCR NAAT named Oscar™ read by hybridization-based vis- RAAD) for HIV RNA detection from whole blood. The micro- ual detection in a completely automated and self-contained RAAD consist of a microfluidic paper-based analytical de- system.141 The results of Accula™ are displayed in around 30 vice (μPAD) with vitrified amplification reagents for RT- min but it requires a dock that controls the fluidic and tem- LAMP, thermally actuated valves, and a temperature control peratures of the cassette.136 Video from reference 137 can circuit in a reusable plastic cassette. The RT-LAMP products provide a more clear visualization of the workflow. Accula™ were visualized in a LFIA (Figure 7a). N. M. Rodriguez et also proved that assays for these kinds of diagnostic tests al.144 created a paper fluidic foldable chip for nucleic acid can be rapidly developed, produced and deployed. On Janu- extraction, IA, and LF detection of human papillomavirus ary 23rd, 2020, the company decided to develop an assay (HPV) 16 DNA (Figure 7b). B. H. Park et al.149 proposed a ro- for the Accula™ platform for SARS-CoV-2 detection, and by tatory microfluidic system able to integrate DNA extraction, February 14th they had a working assay with synthetic tar- LAMP and LF results readout (Figure 7c). gets. After performing verification and validation studies, the company submitted for the FDA EUA application, which Lately, paper-based microfluidics and chip-based micro- was granted on March 20, 2020.136 That means a window fluidics have enabled NAAT to be completed in an enclosed time of just two months for developing and deploying an in chamber, which avoids manipulation and cross contamina- vitro diagnostic test for an emerging infectious disease. tion. Advances in dry chemistry to lyophilized reagents eliminate the need for cold-chain transportation and im- The PortNAT® system from Ustar Biotechnologies is an prove stability. The lyophilized enzymes and primers, plus all-in-one test cassette with a reusable incubator which uses the liquid buffers and the readout technology are being in- CAP isothermal amplification.95,102 It has a CE marked tegrated with microfluidics and combined with portable COVID-19 RNA Test that has a TAT of 40 min (35 min of am- heaters or readers for implementing these integrated mo- plification and 5 min for reading the results in the LF lecular LF as a POCT.114,128,144 strip).138,142 Additionally, the not in vain awarded with the Nobel Prize The Veros™ test from Sense Biodetection140 is also for in Chemistry in 2020,150 Clustered Regularly Interspaced COVID-19 diagnosis and has received CE mark. The test is Short Palindromic Repeats (CRISPR) technique, has been based on a patented exponential IA104 and NA detection applied to diagnostic testing.151,152 CRISPR is known as the methods,143 all self-contained in a single-use cassette with a “next-generation molecular diagnostic technology” because TAT of about 15 min. The test has an electrochemical de- of its advantages such as easy design and specific identifica- sign, lyophilized chemicals and carbon-based readout parti- tion, thus it is a natural next step to use the LF platform as a cles for the LF strip, but needs to be powered with a 1.5 V readout to bring this highly specific and sensitive technique direct-current battery. to the POCT.49 CRISPR/Cas relies on pre-amplification of In agreement with this trend, several LF tests for detec- target sequences, which are next detected by Cas proteins. tion of amplified nucleic acid products have been commer- Combining the CRISPR/Cas system with an IA technique can cialized in the last years, including AmpliVue (Quidel), Amo- improve the sensitivity and solve the specificity issues of dia EasyFLow® (Amodia Bioservice), HybriDetect (Milenia the IA.79,153 The Specific High-sensitivity Enzymatic Re- Biotec), and DCNovations NALF kit (DC Diagnostics).49 porter UnLOCKing (SHERLOCK) and the DNA Endonucle- These tests are used as a readout for nucleic acid amplifica- ase-Targeted CRISPR Trans Reporter (DETECTR) system tion assays of the corresponding company or as a readout are typical combinations of CRISPR and IA. Many assays Figure 7: a) 1) Schematic of the workflow of the microRAAD for HIV testing. 2) Photo of microRAAD connected to phone to power the temperature control circuit. Reproduced with permission from Phillips, E. A.; Moehling, T. J.; Ejendal, K. F. K.; Hoilett, O. S.; Byers, K. M.; Basing, L. A.; Jankowski, L. A.; Bennett, J. B.; Lin, L. K.; Stanciu, L. A.; Linnes, J. C. Microfluidic Rapid and Autonomous Analytical Device (MicroRAAD) to Detect HIV from Whole Blood Samples. Lab Chip 2019, 19 (20), 3375–3386 (ref. 148) Copyright © (2019) the Royal Society of Chemistry. b) 1) Image of the paper fluidic chip. 2) Drawings and dimensions for the adhesive base of the chip. 3) Schematic of chip fabrication steps. Reproduced with permission from Rodriguez, N. M.; Wong, W. S.; Liu, L.; Dewar, R.; Klapperich, C. M. A Fully Integrated Paperfluidic Molecular Diagnostic Chip for the Extraction, Amplification, and Detection of Nucleic Acids from Clinical Samples. Lab Chip 2016, 16 (4), 753–763. (ref. 144). Copyright © (2016) Royal Society of Chemistry. c) 1) A digital image of the rotary device. 2) Schematic of the integrated rotary microdevice for DNA extraction, LAMP, and LF strip detection. 3) (i) Schematic design of the solid phase DNA extraction with microbeads, (ii) Schematic image of the LAMP amplification (iii) Schematics of the LF strip with streptavidin coated AuNPs as a reporter. Reprinted from Park, B. H.; Oh, S. J.; Jung, J. H.; Choi, G.; Seo, J. H.; Kim, D. H.; Lee, E. Y.; Seo, T. S. An Integrated Rotary Microfluidic System with DNA Extraction, Loop-Mediated Isothermal Amplification, and Lateral Flow Strip Based Detection for Point-of-Care Pathogen Diagnostics. Biosens. Bioelectron. 2017, 91, 334–340 (ref. 149). Copyright © (2017) Elsevier. have been developed based on the SHERLOCK/DETECTR Author Contributions method with IA such as RPA, LAMP or RCA, being the results The manuscript was written through contributions of all au- interpreted by different signal output methods, including thors. All authors have given approval to the final version of the LF.153,154 It is expected that the union of these combined manuscript. technologies could be applied in the field of POCT diagnosis in the future. Notes The authors work for or collaborate with Paperdrop Diagnos- CONCLUSIONS tics, a for-profit small-medium enterprise. However, all the au- In addition to being cheap and easy to manufacture, LF thors declare neutrality, objectivity, and no conflicts of interest assays (nowadays the most known type of RDT) are rapid, with the publication of this review. user-friendly, and require no equipment nor battery to work. Although questioned by their sensitivity, they are Biographies suitable for POCT, at-home testing, high-frequency testing, Ana Rubio-Monterde works as an R&D Scientist at Paper- low-resource settings, and emergency use. On the other drop Diagnostics, where she is an Industrial PhD candidate, hand, PCR is a powerful technique used to produce multiple and her thesis is being directed by Prof. Dr. Arben Merkoçi copies of a nucleic acid region of interest, but eliminating and Dr. Daniel Quesada-González. She is a Biologist special- the need of thermocyclers by means of IA methods, freeing ized in Health Biology by Universitat Autonoma de Barce- amplification technologies of energy-demanding and tech- lona and she obtained her MSc in Clinical Analysis Labora- nologically complex systems, opens the door also to POCT tory by Universitat Pompeu Fabra in 2014. She has experi- and in-field applications. Consequently, the application and ence in clinical laboratories in the areas of microbiology, progresses of IA methods are growing, in a way that in the molecular biology, and serology, as well as in the develop- near future IA could rival PCR. ment and verification of in vitro diagnostic tests for CE Shyly, new medical devices that combine the accuracy marking and FDA approval (510K). Her research focuses on and sensitivity of a PCR with the speed and ease of RDT are the design and optimization of molecular rapid diagnostic emerging, allowing to overcome the shortcomings of both tests on paper for antimicrobial resistance determinants techniques and bringing together the best of the two meth- and emerging diseases. ods. However, technical maturity of the IA methods needs to be achieved and, solutions for the unspecific products Daniel Quesada-González, PhD in Chemistry, master in must be found. In addition, advances in microfluids are ris- Nanotechnology and Materials Science, graduated in Chem- ing to the occasion to integrate both in a single device, al- istry. Daniel is the Head of Product Development at Paper- lowing to take the integrated molecular LF test to the POC. drop Diagnostics, a small-medium enterprise in which he Lastly, the inclusion of nanomaterials (rather than has been working since its inception. He has more than 10 AuNPs) in the LF platforms allows achieving lower LOD and years of experience in the development of LFTs, having pub- their properties grant new applications. However, these ad- lished 5 reviews that cover this topic in the last years. Since vances in nanomaterials are still not being reflected in the 2017 he has received more than 1300 citations. Also, he has commercial tests, as the vast majority uses still AuNPs or la- experience in the synthesis, characterization, and conjuga- tex beads. Could be that some limitations still need to be tion of nanomaterials, especially for their application in LF overcome, such as large-scale production or reproducibil- tests. ity/repeatability of the fabrication due to the very complex assays/platforms. Arben Merkoçi Prof. Arben Merkoçi is an ICREA Research Professor and AUTHOR INFORMATION leader of the ICN2 Nanobioelectronics and Biosensors Group. He is also the Co-Editor in Chief of the Biosensors Corresponding Authors and Bioelectronics journal and member of the editorial Daniel Quesada-González - Paperdrop Diagnostics S.L., MRB, board of Electroanalysis, Microchimica Acta. Prof. Merkoçi Campus UAB, 08193 Bellaterra, Spain; orcid.org/0000-0003- has published 319 articles (H-index/citations: Google 3064-2146; Email: dquesada@paperdropdx.com Scholar 87/25890; WOS 70/17682) and supervised 35 Ph.D. theses. He serves also as a scientific evaluator and Arben Merkoçi - Paperdrop Diagnostics S.L., MRB, Campus member of panels of experts of various international gov- UAB, 08193 Bellaterra, Spain; Nanobioelectronics and Biosen- ernmental and nongovernmental agencies (EU-FP and EU- sors Group, Catalan Institute of Nanoscience and Nanotechnol- ERC panels and other panels in Europe, USA, and other ogy (ICN2), CSIC, Barcelona, Spain; The Barcelona Institute of countries) and is the cofounder of two spin-off companies: Science and Technology (BIST), Campus UAB, 08036 Bel- laterra, Barcelona, Spain; ICREA, Institució Catalana de Recerca GraphenicaLab, devoted to graphene patterning, and Paper- i Estudis Avançats, Pg. Lluis Companys 23, 08010, Barcelona, drop Diagnostics, dedicated to clinical diagnostics. Spain; orcid.org/0000-0003-2486-8085 Email: ar- ben.merkoci@icn2.cat ACKNOWLEDGMENT We acknowledge the funding received from Ministerio de Cien- Present Addresses cia e Innovación (Madrid, Spain), which included grants PTQ2018-010113 “IRON” and DIN2018-010115 “LáMiNa”. †Daniel Quesada-González - Nanobioelectronics and Biosen- ICN2 is funded by CERCA programme, Generalitat de Catalu- sors Group, Catalan Institute of Nanoscience and Nanotechnol- nya. Grant SEV-2017-0706 funded by ogy (ICN2), CSIC, Barcelona, Spain; orcid.org/0000-0003- MCIN/AEI/10.13039/501100011033. Grant PID2021- 3064-2146; Email: daniel.quesada@icn2.cat 124795NB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” is also acknowledged. A.R.M. acknowledges https://doi.org/10.1016/S0140-6736(21)02346-1. Universitat Autonoma de Barcelona (UAB) for the possibility of (15) Cubas-Atienzar, A. 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(145) BUSER, J.; BISHOP, J.; GUELIG, D.; HOWELL, A.; KUMAR, S.; YAGER, P.; ABE, K.; Erin HEINIGER; BYRNES, S.; ANDERSON, C.; KAUFFMAN, P. C.; WHEELER, M. Lateral Flow-Based Systems and Methods. US 2021/0349087 A1, 2021. Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for TOC graphic specifications. Insert Table of Contents artwork here The author generated this image with DALL·E, OpenAI’s system that generates original images and art- work from a natural language description. 26