pubs.acs.org/CR Review Toward Next Generation Lateral Flow Assays: Integration of Nanomaterials Amadeo Sena-Torralba, Ruslan Á lvarez-Diduk, Claudio Parolo, Andrew Piper, and Arben Merkoçi* Cite This: Chem. Rev. 2022, 122, 14881−14910 Read Online ACCESS Metrics & More Article Recommendations ABSTRACT: Lateral flow assays (LFAs) are currently the most used point-of-care sensors for both diagnostic (e.g., pregnancy test, COVID-19 monitoring) and environmental (e.g., pesticides and bacterial monitoring) applications. Although the core of LFA technology was developed several decades ago, in recent years the integration of novel nanomaterials as signal transducers or receptor immobilization platforms has brought improved analytical capabilities. In this Review, we present how nanomaterial-based LFAs can address the inherent challenges of point-of-care (PoC) diagnostics such as sensitivity enhancement, lowering of detection limits, multiplexing, and quantification of analytes in complex samples. Specifically, we highlight the strategies that can synergistically solve the limitations of current LFAs and that have proven commercial feasibility. Finally, we discuss the barriers toward commercialization and the next generation of LFAs. CONTENTS 5.2. Sample Pretreatments Based on Active yet Automatic Manipulation of the Sample 14900 1. Introduction 14881 5.3. Bioreceptor Engineering 14900 2. Quantification at the PoC 14882 6. LFA Validation and Barriers toward Commercial- 2.1. LFA Semiquantification by Naked Eye 14882 ization 14901 2.2. Smartphone-Based Readers 14883 7. Outlook 14902 2.3. Portable LFA Readers for Alternative Signal Author Information 14903 Transduction Methods 14884 Corresponding Author 14903 2.3.1. Magnetic Readers 14884 Authors 14903 2.3.2. Thermal Contrast Readers 14885 Notes 14903 2.3.3. Surface-Enhanced Raman Scattering Biographies 14903 Readers 14886 Acknowledgments 14904 3. Sensitivity Enhancement 14886 List of Abbreviations 14904 3 .1. Modulation of the Flow Dynamics 14887 References 14905 3.2. Sample Preconcentration 14890 3.3. Increasing Bioreceptor Concentration 14890 3.4. Novel Signal Transducers 14890 3.5. Signal Amplification 14891 1. INTRODUCTION 3.6. Alternative Signal Transduction Methods 14892 In 2003, the World Health Organization (WHO) published its 4. Multiplexing 14893 ASSURED criteria which outlined the ideal test for universally 4.1. Multiplexing Enabled by Architecture Mod- available diagnostics. These criteria stipulate that point-of-care ifications of the LFA Strip 14894 (PoC) diagnostics should be Affordable, Sensitive, Specific, 4.1.1. Several Detection Lines in Parallel 14894 User-friendly, Rapid and robust, Equipment-free, and Deliver- 4.1.2. Microarrays 14895 4.1.3. Combination of LFA Strips and Design Received: December 7, 2021 of Innovative Paper Configurations 14895 Published: September 6, 2022 4.2. Using Nanoparticles for Multiplexing 14896 4.2.1. Multiplexing in a Single TL 14897 5. Evaluation of Complex Samples 14898 5.1. Sample Filtration 14898 © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.chemrev.1c01012 14881 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Table 1. Comparison of the Readout Systems for LFA Quantification reader signal detection of LoD (pg mL−1) price ($) size (cm3) weight (g) ref 4 −1 smartphone-based colorimetric E. coli O157:H7 10 CFU mL ∼200 2.5 × 3.8 × 2 400 38 smartphone-based colorimetric HIgG 1.00 × 1004 ∼250 1.5 × 0.8 × 0.1 140 39 smartphone-based colorimetric EVD-IgG 2.00 × 1005 ∼200 13.6 × 6.9 × 0.7 130 4 smartphone-based colorimetric HIgG 7.50 × 1004 214 14 × 7 × 1 130 40 smartphone-based luminescence PSA 100 ∼200 41 smartphone-based colorimetric and luminescence AFB1 590 ∼200 7×7×7 42 UCNP reader luminescence triamcinolone acetonide 980 30 × 30 × 15 2000 47 UCNP reader luminescence ST-2 5000 ∼700 24 × 9.4 × 5.4 900 48 UCNP reader luminescence ochratoxin A 3000 ∼650 49 thermal contrast reader TCA hCG 180 ∼500 13.3 × 10.8 × 7.3 58 SERS reader SERS hCG 106 >3000 18 × 11 × 4.7 >1000 59 photoacoustic reader PA glucose 5.40 × 1006 ∼1300 4×2×2 >4000 60 able to end-users. Two additional criteria, R (real-time “yes/no” answer obtained from qualitative LFAs.25 For some connectivity) and E (ease of specimen collection and applications, such as pregnancy tests, a simple binary readout is environmental friendliness), were added then to the original sufficient; the person taking the test is either pregnant or not. ASSURED, creating the new acronym of REASSURED.1 However, assay quantification is vital in applications where the Various PoC diagnostic platforms have been developed to concentration at which the target analyte is present can be used realize biosensors that fulfill these criteria in the nearly two to inform decision making or where changes in concentration decades since they were published. Perhaps none more so than need to be monitored. Such examples include tests for analytes lateral flow assays (LFAs), which have risen to prominence that are present under normal conditions but whose thanks to their low cost and ease of use.2,3 LFAs are currently concentrations can be elevated or decreased in certain the most used PoC sensors for diagnostic4 (e.g., pregnancy conditions. Perhaps the best example of this is a glucose test,5 COVID-19 monitoring6,7), environmental (e.g., pesti- sensor: a binary test saying that glucose is present in a sample cides,8 heavy metals,9 and bacterial monitoring10), and food matrix is not desirable; however, a quantifiable assay that safety applications (e.g., foodborne allergens11 and patho- determines whether the glucose concentration in a sample is gens12).13 The main reason that LFAs are so popular is higher or lower than accepted healthy limits is. There are a because they are made on paper-based substrates. Paper-based multitude of more complex clinical scenarios (e.g., cancer), materials enable low-cost and sustainable manufacturing, while where variations in the levels and ratios of several biomarkers their porous matrices provide equipment-free microfluidics (molecule’s overexpressions and downregulations) give insight (driven by capillary forces), a biocompatible scaffold capable of on the efficacy of therapies and disease progression/ supporting biointeractions (e.g., between antibodies and prognosis.26,27 Similar scenarios exist in food and environ- antigens), and a flexible substrate on which diverse nano- mental safety applications, where, for instance, the slight biosensing designs and strategies can be developed (e.g., increase of food-borne allergenic content or pollutant origami, barriers, and constrictions).14 The fundamentals of concentrations in a river beyond accepted safe limits can LFA have been extensively covered in several peer-reviewed have lethal consequences.11,28 manuscripts and guides.15,16 The potential to quantify the signal on an LFA is determined While LFAs are extremely versatile, they are still limited to by the nature of the signal generated at the test line (TL) the qualitative detection of a single highly concentrated which needs to be in some way related to the concentration of analyte, lacking the sensitivity required for low-concentration biomarker in the sample. The signal obtained must then be analyte detection, especially in complex biological matrices. As compared to a calibration curve obtained by performing the such, much of the recent research on nanoparticle-based LFAs assay with known concentrations of the analyte across the has focused on increasing their sensitivity,17−19 multiplex- relevant concentration range in as similar a sample matrix as ing,20,21 and novel methods to allow measurements in complex possible.29 media,22,23 as well as for detecting new biomarkers. However, Quantification in LFAs has been performed by purpose-built most publications will seek to address each of these issues readers with simple operation procedures. In all cases these individually. To the best of our knowledge this is the first should be designed to be portable, robust, and cheap for on- review that brings together the most recent, cutting-edge site/PoC use. Recent advances have included the development approaches obtained by the scientific community during the of software and apps as well as readers for more complex signal last 10 years, emphasizing the strategies that can synergistically analysis, including using new techniques (e.g., SERS) and solve the inherent challenges of developing PoC LFAs for novel LFA design architectures (e.g., microarrays). clinical applications. This is vital in order to achieve the next In this section, we will review the recent advances in the generation of LFAs. The Review finishes by discussing the development of LFA readers capable of LFA quantification in barriers toward commercialization and where lie the future PoC/on-site scenarios. We compare the operation mode, price, perspectives of the field. dimensions, and reported assay sensitivity for each reader (Table 1) and will give greater emphasis to those that are best 2. QUANTIFICATION AT THE POC suited to commercialization. Quantitative analysis is defined as the ability to define the 2.1. LFA Semiquantification by Naked Eye concentration of an analyte in a sample matrix.24 Its use in Semiquantification relies on determining an approximate LFAs makes the assays much more powerful than the binary quantity of the target analyte by visual inspection, without 14882 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 1. (A, i) Picture of microscope lenses attached to a smartphone camera that were used to photograph the detection area colorimetric LFA strips. (A, ii) Picture of the LFA strips taken with the microscopic lens and the smartphone camera. Reprinted (adapted) with permission from ref 39. Copyright 2019 Elsevier. (B, i) Schematic representation of a 3D-printed cartridge that contains all the optical apparatus for smartphone-based quantification of colorimetric and fluorescent LFAs. (B, ii) Screenshots from the universal detection app, showing the image analysis features for assay quantification. Reprinted (adapted) with permission from ref 42. Copyright 2020 Elsevier. (C, i) Picture of a portable UCNPs-LFA reader with dimensions of 24 × 9.4 × 5.4 cm and 0.9 kg weight. (C, ii) Schematic representation of the evaluation of the strips, in which the laser light is transmitted by the dichroic mirror at 45° to the detection zone. The smartphone camera captures the luminescence emission of the UCNPs present in the test and control lines, while the infrared filter blocks the laser’s residual light. Reprinted (adapted) with permission from ref 48. Copyright 2019 Elsevier. the need for external instrumentation. The methods to make out of the strip and inserted into a sample reservoir at the base an LFA semiquantifiable are to (i) relate a particular signal of a microfluidic ruler. The PtNPs catalyze the generation of intensity or color tonality with a target concentration,30 (ii) oxygen that pushes red ink along the microfluidic channel. The design the assay so that the signal appears only when the distance traveled by the ink within a specific time can be detected target analyte is above a threshold value,31 or (iii) directly related to the concentration of PtNPs captured on the represent different target analyte concentrations using different TL and, therefore, to the concentration of target analyte test lines.32 However, Saiykhan et al. have developed an present in the sample. The authors have applied this approach innovative method that enables the semiquantification of blood for the detection of prostate specific antigen (PSA) in clinical plasma fibrinogen, based on the flow rate and distance traveled samples, achieving a limit of detection (LoD) of 0.54 ng mL−1 by the sample. In this work a wax-printed chromatography and a linear range of 0−12 ng mL−1.34 paper strip was functionalized with bovine thrombin. This 2.2. Smartphone-Based Readers causes the soluble fibrinogen to precipitate as the sample flows along the strip. Thus, the higher the concentration of Rather than developing new hardware for LFA quantification fibrinogen in the sample, the lower the flow rate and distance and analysis, it would be beneficial to use ubiquitously available traveled. The paper strip is placed inside a holder with a built- technologies for assay analysis. Smartphones have revolution- in scale (with millimeter resolution). This enables the visual ized the way we live; their portability, accessibility, widespread monitoring of the distance traveled by the sample. The device use, and sophistication have motivated their use as LFA is simple, portable, and cost-effective, enabling the PoC readout systems for PoC quantification.35 Modern smart- semiquantification of fibrinogen in a range of 0.5 to 7.0 mg phones are an interesting alternative to conventional benchtop mL−1.33 The use of rulers has also been reported by Li et al., readers, since they are portable miniaturized computers with albeit in a much more complex system. The working principle large random access memory, high-speed CPUs, sophisticated of their assay relies on the use of antibody-functionalized camera lenses with Wi-Fi/Bluetooth, and IR network platinum nanoparticles (PtNPs) that are captured on the TL as connectivity.36 Thus, they possess all the features required of in a conventional LFA. After the assay has run, the TL is cut a PoC/on-site device: capable of assay quantification and data 14883 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review storage and transfer.37,36 When taking pictures of an LFA for Upconverting nanoparticles (UCNPs) are luminescent quantification, it is important that the lighting conditions and nanomaterials that display anti-Stokes shifts; i.e., the emitted optical settings of the camera are optimized and kept constant. photon has a higher energy than the incident photon. In such Variations in these factors can cause variations in the measured circumstances, it is possible to irradiate with light in the NIR biomarker concentration. In this section, we will review recent range of the spectrum and measure light emitted in the visible advances in the use of smartphones for LFA quantification. or ultraviolet range.43 UCNPs have several physical properties The use of 3D printers allows the fabrication of mobile that make them suited for LFA quantification such as narrow phone supports that guarantee the correct lighting and distance emission spectra, high chemical stability, long luminescence between the sample and the camera. This enables the lifetimes, and high resistance to photoquenching and photo- reproducible collection of images for quantification.15 For bleaching.44 The evolution of the UCNP reading systems has example, Jung et al. have developed a smartphone-based reader gone from the use of bulky microtiter plate readers45 to the use consisting of a 3D-printed holder, a smartphone, and a of custom-built commercially available fluorescent readers preinstalled dedicated android app. The holder includes a (UCP-Quant)46,47 and, in recent years, the development of smartphone cradle with optical lens that provides a 3× image smartphone-based readers. Gong et al. have developed a amplification and an LED light source with a reflector that compact (24 × 9.4 × 5.4 cm) and ultralight (0.9 kg) static focuses the light into a diffuser that spreads the light evenly image acquisition device (Figure 1Ci). The device consists of a across the strip. Moreover, the android app incorporates data compact infrared laser (exc. at 980 nm), a dichroic mirror analysis software that calculates the concentration of analyte in (with 98% light transmission at 980 nm), and an infrared filter the sample based on a prestored calibration curve. The authors with nearly 0% transmittance at 980 nm (Figure 1Cii). The report an LoD of 104 CFU/mL for the direct detection of image is acquired with a smartphone with an image analysis Escherichia coli O157:H7 in ground beef.38 In another work, app that enables simple user operation and easy result Quesada et al. improved the performance of their smartphone- interpretation. The device has been validated for the detection based readout by attaching a cheap (20 USD) and of nucleic acids, small proteins, heavy metal ions, and bacteria, commercially available microscopic lens onto their smartphone making this UCNPs-LFA reader ideally suited for commerci- camera (Figure 1Ai). This increases the number of pixels that alization.48 can be recorded, improving the quality of high-magnification Similarly, Jin et al. have developed a portable detection images taken of the test area (Figure 1Aii).39 reader based on a 980 nm laser that excites the TL at a 45° Other researchers have exploited other smartphone angle. They have evaluated UCNP-LFA strips with a CCD capabilities. For instance, Brangel et al. have taken advantage camera (Nikon) and a smartphone (and using ImageJ of the smartphone’s GPS technology to develop an app that software). They found that the smartphone was not as enables the geotagging of the samples to aid. They proved the sensitive as the camera but still had a good linear correlation applicability of this feature for Ebola surveillance screening and for ochratoxin A, mercury ions, and salmonella.49 This result is management of infected patients in Uganda.4 Miller et al. have not surprising and highlights the importance of the camera/ used smartphone video analysis as a cost-effective and simple smartphone in these readers, which is often overlooked in method to calculate the dissociation constant (KD) of analyte publications. and bioreceptor in an LFA. The authors suggest that this 2.3. Portable LFA Readers for Alternative Signal strategy can overcome several barriers to the quantitative Transduction Methods analysis of LFAs, such as color inhomogeneity and Optical detection methods using colorimetric or fluorescent reproducibility. The achieved KD showed excellent agreement labels to give a visual readout are by far the most popular type with a reference benchtop interferometer.40 of LFAs. However, despite their simplicity, cost-effectiveness, Smartphone-based readers have also been applied for the and fast acquisition times, these detection methods typically quantification of fluorescent LFAs. Danthanarayana et al. have suffer from poor sensitivities, high noise levels, and low developed a portable imaging instrument for the evaluation of reproducibility.50 As mentioned previously, this has led to the nanophosphor-based LFAs. In this case, the authors used the development of alternative signal transduction methods; flash of the smartphone camera to excite the luminescent however, these alternative methods need portable readers for nanoparticles for 3 s; then, an image is acquired after a 100 ms PoC/on-site assay quantification. In this section, we will review delay. This time-gated imaging is a commonly used strategy to advances in PoC readers for other (nonvisual) transduction decrease the background signal since it allows the applied light methods to decay before the image is acquired. This approach enabled 2.3.1. Magnetic Readers. Magnetic nanoparticles have the detection of PSA and hCG with LoDs of 0.1 and 1 ng/mL, been extensively utilized in LFAs for sample pretreatment, respectively.41 Liu et al. have developed a smartphone-based particularly for the isolation and preconcentration of the reader for the dual quantification of colorimetric and analyte. However, these nanoparticles also offer outstanding fluorescent LFAs. They 3D-printed a 70 mm long cubic box analytical capabilities when used as labels in magnetometric using black photosensitive materials. The cartridge contains detection.51 There are currently two reported ways of both white and UV LEDs as light sources for visible and quantifying magnetic nanoparticles, by inductive sensing and fluorescent signaler excitation (Figure 1Bi). The images taken by magnetic particle quantification (MPQ). Both require a coil with the smartphone camera were analyzed with an Android that excites the magnetic nanoparticles and interrogates the Studio app that splits the image into red, green, and blue magnetic signal generated at the TL of the LFA strip. The channels, with each channel being used to detect a different inductive sensing measures the magnitude and phase of the signaler in a multiplexed LFA (Figure 1Bii). The results coil impedance, which varies depending on the change in obtained showed good consistency when validated against a magnetic permeability of the magnetic nanoparticles.52 In liquid chromatography mass spectrometer (LC-MS).42 MPQ the coil generates a nonlinear magnetic field that 14884 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 2. (A, i) Schematic representation of the MPQ quantification procedure of LFAs using a PoC MPQ reader. Reprinted (adapted) with permission from ref 54. Copyright 2016 Elsevier. (A, ii) Schematic representation of the simultaneous quantification of three LFA strips using a cartridge and the MPQ reader. Reprinted (adapted) with permission from ref 51. Copyright 2016 American Chemical Society. (B) Pictures of a portable LFA reader used for the quantification of the heat generated on the LFA TL due to thermal contrast amplification. The reader includes sensors for the evaluation of both heat conduction and radiation. Reprinted (adapted) with permission from ref 58. Copyright 2016 Springer. (C, i) Schematic representation of a SERS reader. (C, ii) Picture of the SERS reader during the scanning of the LFA strips. Reprinted (adapted) with permission from ref 59. Copyright 2019 Wiley. alternates between two frequencies, f1 and f 2. The signal of antibody−antigen binding kinetics.54 The MPQ reader is emitted by the nanoparticles after their magnetization is also suited to multiplexing strategies, by the use of several coils recorded by a coil attached to a magnetic reader.53 that enable the interrogation of different TLs present on a Magnetic quantification offers several advantages over the single strip55 or by the simultaneous evaluation of several strips traditional colorimetric reading, such as deep signal registration in a cartridge (Figure 2Aii).56 MPQ readers are still in the early (it measures the signal generated in the TL volume rather than development stage with most prototypes only existing in what is observable at the NC surface), extremely wide dynamic research laboratories, and none are commercially available at linear ranges (up to 7 orders of magnitude), and better the time of writing. This makes it difficult to assess the sensitivity when evaluating complex samples (the opacity of commercial viability of such devices. the biological sample does not hinder the magnetic readout). 2.3.2. Thermal Contrast Readers. The use of TCA has Based on these analytical features, some researchers have been widely reported in recent years as a strategy to improve focused on developing portable and cost-effective readers the sensitivity of LFAs. As previously explained in section 2, capable of performing MPQ at the PoC. Orlov et al. have the working principle relies on the light-to-heat transduction of designed an MPQ method and portable reader with out- metallic nanoparticles upon their irradiation with an NIR laser. standing analytical properties. Their assay used 200 nm The cornerstone of this sensing method is the high resolution diameter magneto-radioactive 59Fe-based MPs conjugated to of infrared cameras, which can recognize temperature differ- monoclonal anti-PSA. The assay quantification is performed by ences of just 0.1 °C, providing high assay sensitivity compared the incorporation of the test strip in the measuring coil of the to image analysis of optical readout methods. As was the case MPQ reader (Figure 2Ai). They report an LoD of 25 pg mL−1 with magnetic detection, TCA can overcome the light of PSA in human serum, a dynamic range spanning 3.5 orders scattering issues of colorimetric detection systems. Recent of magnitude, and a high dose−response sensitivity of klog = 3. research has focused on the development of portable The authors also suggest that the developed MPQ reader can alternatives to the current benchtop TCA readers.57 Qu et perform real-time MP mapping during the biorecognition al. have developed the first portable thermal contrast reader. It event. This could be a useful tool for the rapid determination is small (133 × 108 × 73 mm) and has been fabricated with 14885 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Table 2. Comparison of the Sensitivity Enhancement Strategies Reported for LFA strategy method/material detection of enhancement comp. to ref flow rate decrease hydrophobic PCL nanofibers synthetic Zika virus 10-fold conv. LFA 65 flow rate decrease cellulose nanofiber aerogel mouse IgG 1000-fold conv. LFA 66 flow rate decrease and reagents reduce detection area from 5 mm to 1 mm C-reactive protein 30-fold conv. LFA 67 concentration flow rate decrease and wax pillars HIgG 3-fold conv. LFA 69 pseudoturbulence generation internal incubation on TL dissolvable wax barrier HIgG 51.7-fold conv. LFA 70 flow control centrifugal forces PSA 6.2-fold conv. LFA 72 sample preconcentration magnetic focusing valosin-containing protein 4000-fold no preconc. 73 sample preconcentration magnetic focusing influenza A (H1N1) 26-fold no magnetic 74 antigen focusing sample preconcentration filtration E. coli 10-fold no filtration 10 sample preconcentration Nafion-based ICP β-hCG 3.9-fold no preconc. 75 increase antibodies concentration fixing conj. AuNPs in TL C. sakazakii 100-fold conv. LFA 76 increase signal contrast carbon nanoparticles E. Coli 3.8-fold conv. LFA 77 increase signal contrast MWCNTs Meth. 10-fold AuNPs-LFA 78 fluorescence signal transduction and magnetic CdSe/ZnS QDs nanobeads Influenza A LoD of no comparison 81 sample pre-concentration 22 pfu mL−1 ratiometry with target-dependent and red CdSe/ZnS QDs and blue polystyrene H-IgG 78-fold AuNPs-LFA 82 independent reporters nanobeads fluorescence signal transduction and fluorescent nanodiamonds biotin 105-fold AuNPs-LFA 84 increased signal-to-noise ratio signal amplification silver staining of AuNPs Troponin I 10-fold AuNPs-LFA 88 signal amplification AuNPs coated with HRP HIgG 10-fold AuNPs-LFA 89 signal amplification AuNPs coated with ALP potato virus X 10-fold AuNPs-LFA 90 signal amplification AuNPs coated with ultrathin Pt skins PSA 100-fold AuNPs-LFA 91 signal amplification porous platinum core−shell nanocatalysts P24 100-fold no catalysis 92 (PtNCs) alternative signal transduction thermal contrast amplification influenza, malaria, and C. 8-fold AuNPs-LFA 57 dif ficile alternative signal transduction thermal contrast amplification P24 protein LoD of no comparison 97 8 pg mL−1 alternative signal transduction thermal contrast amplification HCG 12-fold AuNPs-LFA 58 alternative signal transduction photoacoustic imaging cryptococcal antigen 100-fold AuNPs-LFA 98 (CrAg) from Cryptococcus alternative signal transduction and surface enhanced Raman scattering and H1N1 and HAdV viruses 2000-fold AuNPs-LFA 103 sample preconcentration magnetic focusing alternative signal transduction and flow surface enhanced Raman scattering and thyroid stimulating LoD of no comparison 104 modulation sequential flow of Raman reporters hormone (TSH) 0.15 μIU/mL alternative signal transduction and surface enhanced Raman scattering and S. Enteritidis no SERS-LFA based 105 bioreceptors bacteriophages as bioreceptors enhancement on antibodies low-cost components (Figure 2B). They validate their reader 2C). This realized a rapid test with data acquisition times with an LFA that uses anti-hCG functionalized Au nanoprisms. several orders of magnitude shorter than conventional Raman Once the LFA strip is placed in the reader, an NIR light beam microscopes, ideal for PoC applications. Moreover, the method is focused on the TL. The gold nanoprisms absorb the light was 15 times more sensitive than the conventional AuNP- and generate heat, which is detected by either a semiconductor based colorimetric detection of hCG (LoD of 0.16 ng mL−1).59 sensor (heat conduction sensing) or infrared thermometer The main drawback to SERS readers being used in PoC (heat radiation sensing). The semiconductor sensor has a applications is their cost. The most expensive components are resolution of 16 bit (0.0078 °C), while the infrared the fiber optic Raman probes (>$2000) and the portable thermometer has a slightly higher 17 bit resolution (0.0039 spectrometers (>$800). As well as finding ways to reduce the °C). the higher sensitivity of the radiation method did however costs of these components, more powerful and user-friendly make it more prone to noise. The researchers recommend the software will also need to be developed before SERS readout selection of either sensing mode depending on the assay platforms can be used in PoC LFA quantification applications. requirements. This approach yielded a 12-fold lower LoD (180 pg mL−1) than the comparable AuNP-based LFA for hCG 3. SENSITIVITY ENHANCEMENT detection.58 As for any diagnostic device, the optimization and design of an 2.3.3. Surface-Enhanced Raman Scattering Readers. LFA should be tailored toward the intended final use; for There have been several attempts to develop PoC SERS example, it needs to be sensitive across the clinically relevant instruments. For instance, Tran et al. have developed a biomarker range.61 Perhaps the most important analytical portable SERS-LFA reader with a fiber optic probe able to properties are sensitivity (i.e., the ability to discriminate acquire 50 scans at different positions on the TL in 5 s. In this between different analyte concentrations) and limit of work the strip was scanned orthogonally to the TL, and the detection (i.e., the lowest analyte concentration that can be motorized stage would move one step every 10 ms (Figure discriminated from the blank).62 The vast majority of clinical 14886 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 3. (A, i) Schematic representation of a sensitivity enhancement approach based on the introduction of a cellulose nanofiber (CNF) aerogel after the conjugate pad. (A, ii) SEM image of the CNF aerogel, showing an average pore size of 250 μm. (A, iii) Comparison of the flow after 30, 60, and 90 s in strips with and without the CNF aerogel. Reprinted (adapted) with permission from ref 66. Copyright 2021 Elsevier. (B, i) Schematic representation of a sensitivity enhancement approach based on the incorporation of a dissolvable wax barrier after the test line. (B, ii) Optical microscopy picture (40×) of wax barriers with different widths. (B, iii) Picture of the LFA strips with and without the wax barrier, after the detection of HIgG (0−1000 ng mL−1). Reprinted (adapted) with permission from ref 70. Copyright 2020 Elsevier. (C, i) Modulation of the direction of flow of graphene quantum dots by the adjustment of the electrolyte solution pH in a paper-based electrophoretic bioassay. (C, ii) Separation of a mixture of CdSe@ZnS QDs, CdTe QDs, and N,S-doped carbon dots according to the electrophoretic mobility of the nanomaterials. Reprinted (adapted) with permission from ref 71. Copyright 2021 American Chemical Society. applications require the detection of sub-picomolar concen- incubated on the test line (TL) to increase antigen capture, trations of biomarkers. In this section, we present recent and therefore improve the sensitivity of the assay. There are advances aimed at improving the LFA sensitivity when using two main methods of reducing the flow rate in LFAs: the first the noncompetitive assay format (Table 2); we discuss their is by chemically modifying the nitrocellulose NC to reduce the pros and cons as well as their commercialization feasibility. flow rate, and the second is to change the geometry of the LFA 3 .1. Modulation of the Flow Dynamics components. One of the main reasons for the poor sensitivity in LFAs, when The earliest examples of chemical modification of LFAs to compared to other analytical techniques like enzyme-linked slow the flow rate involved the incorporation of salt barriers to immunosorbent assays (ELISA), is that they do not allow as impede the flow. These can be easily drop cast onto the much time for the bioreceptors (probes) to bind to the nitrocellulose and will halt the flow until they have been antigens (targets).63 This is due to the capillary force driven dissolved by the sample solution. Usually these are placed after wicking properties of the paper-based LFA strip, which the test and control lines, but a recent work by Prof. Feng Xu produces a unidirectional flow of the sample at a constant and co-workers stands out because the salt barrier was placed rate through the substrate. Ideally the sample would be in front of the test and control lines.64 Besides the reduction of 14887 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 4. (A, i) Schematic representation of a sensitivity enhancement approach based on sample preconcentration by magnetic focusing. (A, ii, a) TEM image, (b) HAADF-STEM image, and (c−f) EDS elemental mapping images of Pt−P2VP@SPION. Reprinted (adapted) with permission from ref 74. Copyright 2021 American Chemical Society. (B, i) Schematic representation of a sensitivity enhancement approach based on sample filtration. (B, ii) AuNP-based LFA approach for the detection of E. coli in tap water (0−109 CFU mL−1). Reprinted (adapted) with permission from ref 10. Copyright 2021 The Royal Society of Chemistry. the flow velocity and the consequent 10-fold sensitivity on the incorporation of novel materials, but on the reduction enhancement, the authors also claim that the increase in salt of the Test line width from 5 to 1 mm.67 They use a laser to concentration improves the hybridization efficiency of the polymerize a pattern on the nitrocellulose membrane. By nucleic acid bioreceptors used. While this is a low-cost and narrowing the flow path, they achieve an increase in both the simple approach, the high ionic strength of the sample solution flow time and a concentration of the reagents. They report a at the target line will not be compatible with other bioreceptors 30-fold sensitivity enhancement for the detection of C-reactive and can denature some protein and aptameric receptors; protein compared to the assay with no flow focusing. The therefore, its suitability should be evaluated for any given authors point out that this method is low-cost, since the application. polymerization can be performed using conventional lasers Recent advances in the field have focused on using which is advantageous compared to other patterning polymeric chemical barriers as opposed to salts. For example, techniques such as photolithography.67 Yew et al. have electrospun a 10% solution of polycaprolactone Other works have also investigated changing the flow nanofibers onto a nitrocellulose membrane for 60 s in order to geometry to improve LFA sensitivity; for example, Parolo et al. delay the flow time by 17 s (0.29 mm/s). compared to have enlarged the width of the sample and conjugate pads 3- uncoated nitrocellulose (NC) membranes (0.35 mm/s). The fold in order to increase the bed volume of the strip. The incorporation of polycaprolactone increases the hydrophobic- authors achieve an 8-fold enhancement in the limit of ity of the coated region, reducing the flow rate and increasing quantification (LoQ) for HIgG detection; the simplicity of the interaction time between the biorecognition element and this strategy makes it ideal for incorporation into other LFAs.68 the analyte.65 This strategy resulted in a 10-fold increase in the Complex microfluidics can be cheaply, quickly, and easily sensitivity of the assay, compared to the conventional LFA. In introduced into LFAs, using wax printers, to improve the assay such approaches it is important to consider the properties of performance. For example, Rivas et al. introduced wax pillars at the materials being incorporated into the strip, as these should the beginning of the nitrocellulose membrane to produce a in no way compromise the test performance. Tang et al. have flow delay and generate pseudoturbulences in the capillary shown that the insertion of a nanofibrillated cellulose aerogel flow, both of which improve probe−analyte binding. Their (pore size of 100 nm in the wet state) just after the conjugate method resulted in a 3-fold sensitivity enhancement for HIgG pad can decrease the sample flow rate in the lateral flow device detection, in comparison to the conventional LFA.69 The (Figure 3A). The aerogel transforms into a hydrogel-like state position of the printed microfluidics along the nitrocellulose upon wetting, with an almost 103-fold pore size reduction membrane affects the assay’s sensitivity. The incorporation of compared to other reported stacking pads. This results in a the wax pillars just after the conjugate pad (as in the case of 40−60% higher reaction time between the bioreceptors and Rivas et al.) enhances mainly the interaction time of the first the target analyte, which enables a 1000-fold sensitivity biorecognition event, the binding of the target analyte, and the enhancement compared to the LFA without the aerogel labeled bioreceptors, while the incorporation of such artifacts (LoD of 0.72 ng mL−1 for mouse IgG in human serum). It is after the TL provides an increase in the interaction time of noteworthy that the aerogel has a shelf life of 6 months.66 both the first and second biorecognition event, the second Alternatively, Katis et al. have developed a strategy not based being the immuno-sandwich formation with the secondary 14888 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 5. (A, i) TEM image of carbon nanoparticles that form aggregates of 10−100 nm. (A, ii) Photograph of LFA strips after detecting E. coli (0−7.09 pg μL−1) using carbon nanoparticles (black) and AuNPs (red). Reprinted (adapted) with permission from ref 77. Copyright 2021 MDPI. (B, i) TEM image of the magnetic QD nanobeads. (B, ii) Picture of the nanobeads before and after the magnetic enrichment step. (B, iii) Picture of the LFA strips after the detection of H1N1 (0−106 pfu mL−1), showing an LoD of 500 pfu mL−1 by naked eye. Reprinted (adapted) with permission from ref 81. Copyright 2021 Elsevier. (C, i) Color palette generated in the TL of the LFA upon the ratiometric combination of different concentrations of 650-QDs (target-dependent) and 450-NBs (target-independent) reporters. (C, ii) Calibration curve for HIgG detection using LFA strips with increasing concentrations (0−0.1%) of 450-NBS in TL. Reprinted (adapted) with permission from ref 82. Copyright 2021 Wiley. (D, i) Schematic representation of a nanodiamond-based LFA for the detection of HIV-1 RNA. (D, ii) An omega-shaped stripline resonator is fixed under the LFA strip to selectively separate the fluorescence signal at the TL from the background autofluorescence. Reprinted (adapted) with permission from ref 84. Copyright 2020 Springer Nature. antibody. By optimizing both binding events, Sena-Torralba et A novel way of modulating the flow dynamics in LFAs is the al. report a 51.7-fold sensitivity enhancement for HIgG application of alternative mobility forces, such as centrifugal or electrical forces. These provide the end-user with greater detection by inserting a 0.05 mm wide dissolvable wax barrier control of the flow rate and direction of the sample compared 1 mm downstream of the TL (Figure 3B).70 to capillarity, which is governed solely by the bed volume of 14889 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review the strip.71 To this end, Shen et al. report a centrifugal force- usually around 80−100 μg cm−2.16 Researchers are developing assisted LFA, with a constant rotation speed of 1500 rpm, novel strategies that use nanomaterials with high surface-to- adjusted by a portable dedicated device.72 This test enabled a volume ratios to increase the immobilization density of 6.2-fold sensitivity enhancement for prostate specific antigen bioreceptors. For instance, Pan et al. used immobilized (PSA) detection in human serum.72 Likewise, Sena-Torralba et AuNPs functionalized with antibodies to the test line. al. have developed an LFA that incorporates electrophoresis. In Simultaneously increasing the surface area and thereby this way, the flow rate and direction of the sample can be antibody concentration at the test line, and the pre-existence precisely adjusted by the regulation of the voltage applied to, of AuNP at the test line, meant the signal from non- and pH of, the electrolyte solution (Figure 3Ci). The authors immobilized AuNPs from the conjugate pad was evident at prove that this paper-based electrophoretic bioassay can lower target concentrations. With this method they were able separate a mixture of quantum dots of different sizes and to immobilize up to 2.2 mg mL−1 of antibodies at the TL and charges in just 10 min, due to the differences in the achieve a 100-fold sensitivity enhancement for the detection of electrophoretic mobility of both nanomaterials (Figure Cronobacter sakazakii compared to the conventional LFA.76 In 3Cii).71 Based on this proof of concept, the researchers are this kind of approach, the nanomaterial selection dictates the currently working on the application of this approach for the assay’s performance. The nanomaterial at the TL needs to have betterment of the LFA sensitivity. the following four properties: (1) It needs to be small enough 3.2. Sample Preconcentration to penetrate the pores of the nitrocellulose membrane. (2) The suspension should avoid coffee ring effects. (3) The nanoma- The sensitivity of LFAs can be vastly improved by the terial should contain functional groups that enable the oriented preconcentration/purification of the target analyte present in conjugation of the antibodies. (4) The optical properties of the the sample, which is usually present at low levels and in a nanomaterial should not interfere with the detection signal. sample matrix with other biomolecules capable of interfering with the assay. Sample enrichment can be performed using 3.4. Novel Signal Transducers magnetic separation or filtration. The former has been applied Most commercial LFAs rely on the use of low-cost colorimetric by Ren et al.,73 who have used antibody-functionalized AuNPs or dyed beads that quickly generate an optical signal magnetic particles in order to concentrate valosin containing that can be directly inspected by the naked eye. However, protein up to 10-fold, yielding a 4000-times greater sensitivity these are not suitable for scenarios where the target analyte is than the conventional LFA.73 Similarly, Son Le et al.74 have present at ultralow concentrations, mainly due to their low used superparamagnetic iron oxide nanoparticles (SPIONs) molar absorptivity, which means that a large accumulation of that provide a magnetic enrichment factor (φ) = 40, for the nanoparticles is required on the TL to generate a measurable detection of C-reactive protein. This enables a 26-fold lower optical signal. An alternative method of enhancing the LoD (0.08 ng mL−1) compared to the conventional AuNP- sensitivity of LFAs is to use signal transducers with higher based LFA (Figure 4A).74 Magnetic enrichment strategies have absorbances that create a stronger contrast with the back- shown great performance in the lab over the past decade; ground signal. Black carbon-based materials have garnered however, their commercialization has been hindered by the fact interest for this application. Porras et al. have reported a 3.8- that they usually require multiple washing steps that make the fold lower LoD when using carbon nanoparticles compared to assay less user-friendly and more prone to error. AuNPs, allowing the detection of E. coli in the nanomolar In the case of sample filtration, its use is limited to range by naked eye (Figure 5A).77 Similarly, Sun et al. have applications with large sample volumes, and only a reported that the use of multiwalled carbon nanotubes representative amount of the sample is evaluated: for instance, (MWCNTs) in LFAs can provide a 10-fold sensitivity the detection of E. coli in tap water. In this regard, Bergua et al. enhancement for methamphetamine detection compared to have developed a filtration system that uses a 0.25 μm pore size conventional AuNP-based LFAs.78 As well as providing an filter, a small peristaltic pump, and microfluidic tubes to filter easily identifiable color change against the white background of 300 mL of water in 15 min. With this approach, the authors the nitrocellulose, this nanomaterial possesses more binding have improved the LoD by 1 order of magnitude to 104 CFU sites than the AuNPs due to its greater surface area. mL−1 (Figure 4B).10 While this is an effective strategy, sample Over the past decade, fluorescence detection has become a filtration relies on the use of external instrumentation and a common way of enhancing the sensitivity of LFAs. Quantum time-consuming pretreatment of the sample. It is beneficial to dots (QDs) are among the most used labels due to their integrate a sample preconcentration step into the LFA strip. narrow emission peaks and high quantum yield (high photon Lee et al. have developed an LFA with a Nafion-based ion emission rate).79 In such systems it is possible to measure concentration polarization (ICP) preconcentrator integrated in either the absorbance or the emission of the TL to calculate the conjugate pad. ICP is an ion transport phenomenon that the analyte binding concentration; however, the emitted light occurs when ions selectively pass through an ion exchange membrane. It enables a 15-fold sample preconcentration in just is typically measured. The reason for this is that fluorescence 8 min using a portable 9 V battery.75 With this approach they measurements are more sensitive, mainly due to the way they achieved a 26% sensitivity enhancement for the detection of β- are measured. While absorbance is measured over a bright hCG. background, fluorescence is measured without any reference beam and over a dark background.80 This enables the 3.3. Increasing Bioreceptor Concentration detection of low levels of light. Recently, Bai et al. have One of the most straightforward ways to improve the LFA’s developed magnetic QD nanobeads by forming MnFe2O4 sensitivity is to increase the concentration of the capture magnetic beads that they then coated with PEI. These bioreceptor in the TL, as this will lead to a greater analyte positively charge nanoparticles with magnetic cores were binding efficiency. However, this approach is limited by the coated with CdSe/ZnS to create 200 nm diameter super- loading capacity of the nitrocellulose membranes, which is paramagnetic MnFe2O4 core nanoparticles with “quantum dot” 14890 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 6. (A) Schematic representation of the signal amplification strategy using water-soluble nanofibers and the silver enhancement reaction. Reprinted (adapted) with permission from ref 88. Copyright 2018 Elsevier. (B) Signal amplification strategy using AuNPs coated with Pt ultrathin layers (Au@Pt4L), for the detection of PSA. Reprinted (adapted) with permission from ref 91. Copyright 2017 American Chemical Society. coated surfaces. The multifunctional composite nanoparticles Additionally, they demonstrate the clinical utility of this were shown to be capable of the enrichment and ultrasensitive strategy by proving the detection of HIV-1 RNA in real plasma detection of influenza A virions, capable of detecting as little as samples after 10 min of isothermal amplification.84 Despite the 22 pfu mL−1 in nasopharyngeal swabs (Figure 5B).81 Sena- recent advances in fluorescent LFA sensors, few have been Torralba et al. have developed a simple and cost-effective commercialized, perhaps because they require instrumentation strategy based on the ratiometric combination of target- that conventional LFAs do not (excitation source, dark dependent red CdSe@ZnS QDs (650-QDs) and target- illumination, photodetector, etc.). To address this, portable independent blue polystyrene nanobeads (450-NBs) in LFA versions of this equipment is being developed: such as phone- (Figure 5Ci,ii). The approach enables the precise modulation based fluorometers85 or affordable infrared laser diodes for the of the assay’s dynamic range and sensitivity upon the use of excitation of upconverting nanoparticles (UCNPs) (section different concentrations of both reporters. The authors prove a 2).86 78-fold higher sensitivity than the AuNP-based LFA when 3.5. Signal Amplification detecting HIgG and an 18-fold faster assay time (compared to ELISA).82 As sensitive as fluorescence-based detection Signal amplification is also a well-known strategy for sensitivity methods are, their performance can be hindered by the enhancement in LFA. There are several ways to amplify the intrinsic background fluorescence of nitrocellulose membranes. signal at the test line, the most widely reported of which are The choice of the optimal substrate (nitrocellulose, glass, etc.) AuNP enlargement through silver staining, or coating the when developing an LFA for an analyte depends on many of AuNPs with enzymes or catalytic metals.87 Silver staining has the material, chemical, and optical properties of the substrate, been applied to the detection of Troponin I by the integration which must be tuned for the optical mechanism being used. of water-soluble hybrid nanofibers between the conjugate pad For example, a substrate should be chosen that does not and the TL. Once the AuNPs are bound at the TL, the silver autofluoresce at the same wavelength as the desired enhancement reagents are released from the nanofibers and are fluorophore emits. A recent work by Shah and Yager sets out reduced to metallic silver around the AuNPs (Figure 6A). The a quantitative framework to aid the selection of the appropriate darkening of the TL enables a 10-fold sensitivity enhance- substrate for LFA development.83 ment.88 The integration of the signal amplification reagents in Other alternative fluorophores have also shown promise the LFA strip is the most interesting advancement of this recently, most notably the use of fluorescent nanodiamonds by approach, since the speed and simplicity of use of the LFA is Miller et al. The emission intensity of these highly fluorescent not compromised, but the signal is enhanced. nanoparticles is very high with emissions in the visible Enzymatic signal enhancement typically uses horseradish spectrum at (675 and 550 nm). The researchers have peroxidase (HRP) or alkaline phosphatase (ALP). These incorporated an omega-shaped stripline resonator under the enzymes are traditionally used in ELISA and are commercially LFA strip, which is used to apply a microwave-frequency available, stable, and cheap. Parolo et al. achieved a 1 order of electromagnetic field that selectively modulates the fluores- magnitude sensitivity enhancement in the detection of HIgG cence signal of the nanodiamonds at a set frequency (Figure by the conjugation of AuNPs with anti-HIgG and HRP. The 5Di,ii). With this approach, the authors achieve sub-attomolar HRP catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine (8 × 10−19 M) limits of detection in a model biotin LFA, 105 (TMB) was found to induce the best color change when times more sensitive than the detection obtained with AuNPs. compared to other methods, as would be expected.89 14891 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 7. (A, i) Schematic representation of a dual-flow LFA fabricated using 5 wax patterned nitrocellulose membrane layers. (A, ii) 25 and 45 nm AuNPs are introduced into the first and second inlet, respectively. This sequential addition of differently sized AuNPs allows for reproducible SERS signal enhancement on the TL. Reprinted (adapted) with permission from ref 104. Copyright 2021 American Chemical Society. (B, i) A bacteriophage covalently coupled to AuNPs by carbodiimide coupling is used as a highly specific bioreceptor. (B, ii) The Bacteriophage LFA system enables the quantitative detection of S. enteritidis using SERS. Reprinted (adapted) with permission from ref 105. Copyright 2021 Elsevier. Unfortunately this assay required multiple washing steps, sophisticated detection systems into the simple paper-based which hinders its applicability at the PoC. Moreover, it is architecture of LFAs. The development of PoC readers using reported that the presence of HRP inhibitors or competitors in user-friendly software to simply and quickly obtain and the real sample matrices impedes these LFAs. Panferov et al. interpret assay results is discussed in section 2; in this section, functionalized AuNPs with ALP and obtained a 10-fold we will simply discuss advances in the methodology of different sensitivity enhancement for the detection of potato virus X signal transduction methods. in leaf extracts. The authors claim that the ALP catalyzed LFA Thermal contrast amplification (TCA) is based on the retains all the advantages of conventional LFAs: 15 min assay measurement of the temperature change with an infrared time, no additional equipment, no extra washing steps, and all camera, when irradiating the metal nanoparticles present on the components able to be stored in the dry state.90 the test line with a near-infrared laser. The metal nanoparticles The signal amplification can also be performed by catalytic are used as light-to-heat transducers by adjusting the laser metals such as Pt, thin layers of which can be coated on wavelength within the localized surface plasmon resonance AuNPs. Pt has a peroxidase-like catalytic activity toward the (LSPR) peak of the nanoparticles. With this signal trans- oxidation of TMB, so it can generate a blue TL. This approach duction principle and the relatively high resolution of infrared enables a 100 times sensitivity enhancement for the detection cameras (0.1 °C),96 the TCA approach enhances the of PSA, due to the large molar extinction coefficient of the sensitivity while simultaneously allowing precise quantification oxidized TMB (Figure 6B). The authors also investigated the in LFAs. For instance, Wang and co-workers observed an 8- feasibility of the strategy for human plasma sample analysis, fold sensitivity enhancement for the detection of influenza, demonstrating that the complex sample matrix did not impair malaria, and Clostridioides dif ficile, compared to the optical the catalytic activity of the Pt shells.91 Similarly, Loynachan et signal provided by AuNPs.57 Similarly, Zhan et al. achieved an al. have taken advantage of the high catalytic activity of porous LoD of 8 pg/mL for the detection of p24 protein spiked into platinum core−shell nanocatalysts (PtNCs) to achieve an human serum,97 and Qu et al. obtained a 12-fold lower LoD ultralow limit of detection of 0.8 pg mL−1 for p24 protein by for HCG biomarker compared with the visual detection naked eye.92 mode.58 This detection method requires a careful calibration to compensate for background environmental temperature 3.6. Alternative Signal Transduction Methods fluctuations. In recent years, researchers have made efforts to develop Photoacoustic (PA) imaging is based on the measurement of alternative signal transduction methods in LFAs, to address the acoustic waves generated after the irradiation of metallic fact that current optical readers and sensing methods are nanoparticles with a laser. The PA signal transduction method insufficiently sensitive and precise enough for some clinical includes three steps: absorption of the light by the AuNPs, the applications. While these alternative signal transduction conversion of the absorbed energy into heat, and the heat- methods [thermal contrast amplification (TCA), photo- induced thermal expansion of the air that generates pressure acoustic (PA) analysis, and surface-enhanced Raman scattering oscillations.22 PA imaging provides outstanding sensitivity (SERS)] have been successfully utilized in other biosensing compared to optical imaging due to its ability to penetrate platforms,93−95 the goal has been to integrate these deeper into the nitrocellulose membrane. There is also a 14892 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Table 3. Comparison of the Multiplexing Strategies Reported for LFA target sensitivity strategy signal transducer analytes detection of sample enhancement ref multiple TLs SiO2@DQD 2 SARS-Cov-2-related HIgG and HIgM human serum 104-fold 117 multiple TLs AuNPs 3 miRNA-21, miRNA-155, and miRNA-210 human serum no 118 multiple TLs AuNPs 3 Giardia, Cryptosporidium, and Entamoeba human stool no 119 multiple TLs AuNPs 3 HIV, HCV, and HAV antibodies human serum no 121 multiple TLs FAM and ROX fluorophores 13 HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) human cervi- no 122 and 2 labels cal swab multiple dots AuNPs 7 DNA alleles (FY*01, FY*02, FY02N.01, GYPB*03, GYPB*04, JK*01, human whole no 125 and JK*02) related to four blood group SNPs blood microarray AuNPs 4 morphine, amphetamine, methamphetamine, and benzoylecgonine human urine no 126 multiple strips UCP nanoparticles 10 E. coli O157:H7, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. typhi, S. food samples no 128 enteritidis, S. choleraesuis, V. cholera O1, V. cholera O139, and V. parahemolyticus wax-patterned catalytic signal amplification 3 Clostridioides dif ficile toxins A and B, and glutamate dehydrogenase human stool 8-fold 129 multichannel of AuNPs (GDH) multiple strips magnetic nanolabels 3 BoNT-A, BoNT-B, and BoNT-E milk, apple, 1000-fold 56 and orange juices single TL and orange, red, and green AgNPs 3 DENV NS1 protein, YFV NS1 protein, and ZEBOV glycoprotein no 135 3 labels single TL and AgNBA@Au, AgMB@Au, and 3 CK-MB, cTnI, and Myo cardiac biomarkers human serum 100-fold 136 3 labels AgR6G@Au SERS nanotags multiple TLs red and blue latex beads 4 IgG and IgM specific to DENV and CHIKV human whole no 130 and 2 labels blood multiple TLs AgNPs, spherical and desert- 3 casein, ovalbumin, and hazelnut allergenic proteins biscuits no 131 and 3 labels rose-like AuNPs multiple TLs ZrMOF@CdTe NPs 2 heart-type fatty acid binding protein (h-FABP) and cardiac troponin human serum 10-fold 133 and 2 labels (cTnT) multiple TLs lanthanide-doped nanopar- 3 staphylococcal enterotoxins SEG, SEH, and SEI PBS buffer 100-fold 134 ticles (YVO4: Eu 40%) multiple TLs AgNBA@Au SERS NPs 3 Myoglobin, cTn1 & CK-MB human serum 1000-fold 123 multiple TLs AuNPs labelled with mala- 2 Clostridium dif ficile surface layer protein A (SlpA) and toxin B (ToxB) human stool 1000-fold 124 chite green isothiocyanate (MGITC) greater signal-to-noise ratio, due to the input (light) and to bind to streptavidin coatings on 45 nm AuNP that were output (acoustics) being different energy types. To date, there flown over the TL in a secondary flow step. This caused an is only one reported use of PA in an LFA. This was reported by increase in SERS signal through electromagnetic enhancement Zhao et al., who improved the sensitivity 100-fold when effects. The authors note that the enhancement effect increases detecting cryptococcal antigen (CrAg), compared to the visible as the AuNP size increases, and simulations showed that using signal provided by the same AuNPs.98 As suggested by the 45 nm AuNPs in both flow channels would give a greater SERS authors, a barrier to the adoption of this technique might be enhancement. However, the 25 nm AuNPs are more the high price of the PA detector and oscilloscope. Hence, homogeneously distributed on the TL, which reduces the researchers are working on the development of portable and variation between test strips. With this approach, the authors cost-effective alternatives such as replacing the laser with achieve an LoD of 0.15 μIU/mL for the detection of TSH, LEDs. with 0.5 μIU/mL being the threshold value for the diagnosis of The integration of surface-enhanced Raman scattering hyperthyroidism.104 Finally, Ilhan et al. have used SERS signal (SERS) into LFAs has been extensively reported during the transduction in combination with bacteriophages as bio- past decade as a sensitivity enhancement strategy.99−101 It is receptors. Bacteriophages are viruses that specifically infect based on the ability of metallic nanoparticles to enhance bacterial species (Figure 7B). The authors propose their use in Raman signals.102 More recently, researchers have focused on LFA as an alternative to antibodies, which are time-consuming further enhancing the analytical properties of LFAs by and costly to produce. The bacteriophage LFA system enabled combining the SERS signal transduction with other strategies. an LoD of 7 CFU/mL and exhibited similar analytical For instance, Wang and colleagues have developed a SERS- performance to an antibody-based LFA for the detection of based LFA using Fe3O4@Ag magnetic nanoparticles loaded Salmonella enteritidis.105 with DTNB dyes for dual magnetic sample preconcentration and SERS signal generation. With this strategy, they have 4. MULTIPLEXING achieved a 2000-fold sensitivity enhancement for the detection Given the complex physiology of many pathological states of H1N1 and HAdV viruses, compared to the conventional (e.g., cancer), which produce multiple different biomarkers, the AuNP-based LFA.103 Kim et al. have applied SERS on a dual- ratios and concentrations of which can be indicative of disease flow LFA, which was fabricated using 3-dimensionally stacked progression,106 diagnostic tests should ideally measure as many layers of wax-patterned nitrocellulose membranes (Figure 7A). clinically relevant biomarkers as possible. Information about The smaller 25 nm AuNPs used in this test were functionalized different biomarkers can inform clinical decisions based on the with anti-TSH antibodies and backfilled with biotinylated BSA needs of individual patients.107 This need is also extended to (TSH, thyroid stimulating hormone). The biotin was then able food, environmental, and safety applications, where the assay’s 14893 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 8. (A, i) Schematic representation of a multiplexed assay based on two consecutive TLs for the detection of IgM and IgG for SARS-Cov-2 spike proteins. (A, ii) Elemental mapping images of the SiO2@DQD used as fluorescent labels in the publication by Wang et al. (A, iii) Picture of the LFA strips after performing the assay for the detection of SARS-cov-2 antibodies, where the red fluorescence signal generated in the M, G, and C lines indicates the presence of IgM (M) or IgG (G), with a positive control (C). Reprinted (adapted) with permission from ref 117. Copyright 2021 The Royal Society of Chemistry. (B, i) Schematic representation of the mobile phone reader used to quantitatively evaluate multiplexed LFA microarrays. (B, ii) Picture of the algorithmically determined immunoreaction spot layout, where each row corresponds to a different spotting condition (1−7). (B, iii) Heat map of the microarray used to optimize the spot configuration by machine learning. Reprinted (adapted) with permission from ref 127. Copyright 2020 Springer Nature. utility increases with the number of targets detected (e.g., methods that enable PoC and high-throughput multiplexing detecting multiple pollutants at once would speed up the with ultrahigh sensitivity as well as those that are feasible for analysis of water quality). Multiplexing is the simultaneous commercialization. analysis of several analytes in a single sample and has been 4.1. Multiplexing Enabled by Architecture Modifications of investigated for decades given its obvious time and sample the LFA Strip saving potential.108−110 Considering that most diagnostic 4.1.1. Several Detection Lines in Parallel. The most samples are extracted in limited volumes (e.g., blood and obvious and simple way to perform multiplexing in LFA is by nasopharyngeal swab), multiplexing saves sample volume, time, creating multiple different test lines (TLs) on the detection and cost.111,112 zone, with each TL responsible for the biorecognition of a Despite the many potential advantages that multiplexing different analyte. Most multiplexing approaches that adopt this offers, adding any complexity to the system inevitably leads to methodology report the detection of up to three different practical difficulties that must be overcome. When developing target analytes. The limited length of the detection zone of the multiplexed LFAs there a several biological and physical issues strip, usually around 4 cm, limits the number of TLs that can that need to be resolved.20 One major challenge is the be incorporated into the LFA.15 The first TL must be fixed at a simultaneous detection of analytes present in the sample at proper distance from the conjugate pad so as to provide very different concentrations (e.g., fM and μM),113 as sensors enough time for the first biorecognition event to occur are developed and designed to work across a clinically relevant between the conjugated label and the target analyte.70 This range. This has led to the development of cost-effective and distance should be optimized during the development of each simple strategies that allow the modulation of the assay’s LFA but is rarely less than 2 cm. Additionally, the distance dynamic range. Likewise, it is not always easy to resolve the between TLs should be at least 2 mm to allow the simple error- signal from the detection of each of the biomarkers under free readout by eye.115 Coupled with the inclusion of a control investigation, which is usually solved by using different line (CL), there is an obvious limit on the number of TLs that detection methods (e.g., fluorophores) for each biomarker; can be incorporated into a standard LFA. An obvious solution however, the optimization of the rest of the LFA may not be would be to elongate the LFA strip; however, this causes an ideal for both biomarkers. Data handling has also appeared as a exponential increase in assay time; as described by the bottleneck when using multiplexed sensors. This has resulted Washburn equation.116 in an increase in papers in recent years focusing on the Wang et al. have developed a multiplexed and highly development of software for the analysis and interpretation of sensitive fluorescent LFA for the detection of SARS-CoV-2- large data sets in multiplexed diagnostics.114 related IgG and IgM. They fixed secondary antibodies specific In this section, we will review recent advances and to human IgM and IgG on the TL1 and TL2, respectively technologies that have been developed to address the (Figure 8Ai); in the conjugate pad, they had 200 nm diameter aforementioned challenges in multiplexed LFA development. silica-core@dual quantum dot (QD)−shell nanotags (SiO2@ These involve the design of new LFA architectures/ DQD) (Ems. 615 nm, Exc. 365 nm) functionalized with SARS- functionalities or the use of novel signal transduction CoV-2 spike proteins (Figure 8Aii). The efficacy of this mechanisms (Table 3). Attention will be drawn to those multiplexed LFA was established by the simultaneous 14894 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review detection of IgG and IgM from SARS-cov-2 positive and Multiple TLs need not only be used for optical readouts. negative patient serum samples.117 Zheng et al. developed an Zhang et al. have taken advantage of the ultrasensitive signal LFA with three TLs for the simultaneous detection of micro- and a wide linear dynamic range of SERS, to develop a RNA-21, micro-RNA-155, and micro-RNA-210 cancer bio- multiplexed LFA for the detection of myoglobin, cTnI, and markers in human serum. In such tests, the specificity of the CK-MB cardiac biomarkers in human serum. The authors used probe sequences is vital for proper assay function, to prevent SERS nanotags (silver core/gold shell NPs) loaded with Nile false positive results from nonspecific hybridization/binding.118 blue A dye (AgNBA@Au) and a triple TL assay design. The Multiplexed nucleic acid biomarker detection in LFAs has also assay displayed a linear response over the clinically relevant been reported by Crannell et al. for the detection of DNA concentration ranges of the three biomarkers spanning over 6 sequences specific to Giardia, Cryptosporidium, and Entamoeba orders of magnitude. They were able to obtain LoDs of 3.2, protozoa in stool samples, all of which are usually present at 0.4, and 0.5 pg mL−1 for myoglobin, cTnI, and CK-MB, very low concentrations. To improve assay sensitivity, this respectively.123 Similarly, Hassanain et al. have utilized SERS work used an isothermal amplification method (recombinase for both the multiplexed and highly sensitive detection of polymerase amplification, RPA) to generate more of the DNA Clostridium diff icile surface layer protein A (SlpA) and toxin B sequence of interest, achieving an ultralow LoD of 444, 6, and (ToxB). In this work, the authors report the use of a handheld 9 parasites/test for Giardia, Cryptosporidium, and Entamoeba, Raman spectrometer that enables the PoC quantification of the respectively.119 Many isothermal amplification technologies biomarkers down 0.01 pg μL−1.124 exist and are being investigated for use in LFAs; however, to 4.1.2. Microarrays. To make better use of the test area date, most publications do not perform the amplification in the afforded by lateral flow strips, rather than printing test lines, it test strips but rather ex situ prior to running the assay. is possible to make microarrays using test spots. Once example Likewise, it is common for papers to exclude the amplification of this is the work published by Gomez-Martinez et al., who time from the assay time to result, which they often increase designed a 4 × 2 microarray for the simultaneous detection of substantially. 7 DNA alleles (FY*01, FY*02, FY02N.01, GYPB*03, Lee et al. have developed a triple-TL LFA with a 100% GYPB*04, JK*01, and JK*02) for rapid blood group clinical sensitivity and specificity for the detection of antibodies genotyping. The authors spotted 8 capture oligonucleotide specific to HIV, hepatitis A, and hepatitis C antigenic peptides probes on the test area of a single nitrocellulose membrane, in patient sera. They have achieved this by immobilizing with one of these being the positive control. While this “proteinticles” in the TL, which hold the bioreceptors approach successfully enabled a fast blood group genotyping (antigenic peptides) on the nitrocellulose surface in a (total processing time of 1 h), it did require a LATE-PCR homogeneous orientation and conformation. “Proteinticles” preamplification step which increased the assay complexity and are nanoscale protein particles that self-assemble inside cells price, since extra instrumentation and reagents were with constant 3D structure and surface topology.120 In short, required.125 they are made by inserting the genetic sequence of the viral In another work, Taranova et al. managed to deposit up to antigen peptide of interest to the C- or N-terminus of known 32 spots in a microarray format (4 × 8) on the NC membrane. proteins; for instance, in the work cited here by Lee et al., The spots had a diameter of 250 μm and were precisely peptides from three viruses (HIV, HCV, and HAV) are deposited by dispensing 20 nL of the capture reagents with a inserted into the human ferritin heavy chain to create steel pin and program-controlled manipulator. Rather than proteinticles.121 These are then expressed in bacteria to using this approach to detect 32 different analytes, they used produce large protein complexes with structured, orientated, the 8 spots in each row as replicates for the detection of 4 3-dimensional scaffolds containing the antigen peptides being drugs (morphine, amphetamine, methamphetamine, and used as a bioreceptor in the assay. Proteinticles serve as an benzoylecgonine) in urine. Making repeat measurements in a alternative to the direct fixation of the peptides on the TL, single strip increases the robustness of the assay results, which normally suffers from limitations such as uncontrolled allowing for more accurate quantification. The authors claim orientation, clustering, inactivation, or instability of the that with the resolution of their setup, they have the capability peptides, which can reduce the accuracy of the assay.121 to print arrays with up to 150 spots in the detection zone.126 The simplest method of multiplexing without extending the This would be a large step forward in the development of high- LFA strip length is to print test lines with two different throughput multiplexed LFAs. Ballard et al. have developed a bioreceptors that conjugate to analytes bound to different machine-learning-based framework to determine the optimal reporters (i.e., fluorophores that emit at different wavelengths). configuration of the spots in the microarray and to quantify the This method can be further multiplexed by printing multiple analyte concentration. A mobile phone-based readout system test lines, as has been shown by Xu et al.122 who have that uses custom-built image processing software to analyze the developed a multiplexed LFA for the simultaneous detection of microarrays has also been developed (Figure 8Bi). The the nucleic acid sequences specific to 13 different human algorithm is designed for assay optimization, designed to papillomavirus (HPV) clades (16, 18, 31, 33, 35, 39, 45, 51, 52, accurately select the best sensing conditions out of up to seven 56, 58, 59, and 68). The researchers deposited 7 TLs, with 2 possibilities being tested in the microarray (Figure 8Bii). different bioreceptors in each TL, and used two fluorophores Additionally, the deep-learning framework developed enables (FAM and ROX) as reporters. In this work, the nucleic acids the selection of the best spatial configuration of the were preamplified by linear-after-the-exponential (LATE)- immobilized spots (Figure 8Biii).127 PCR. As in all assays that preamplify the DNA, the sensitivity 4.1.3. Combination of LFA Strips and Design of is determined by the amplification method; as such, they Innovative Paper Configurations. A quick and simple way report an LoD of 10−102 copies plasmid DNA/μL. They also to develop a multiplexed LFA is by connecting several test report a high specificity with no cross-reactivity among 31 strips to a single sample pad. This has been reported by Zhao common HPV types.122 et al., who have developed a multiplex strategy based on a 10- 14895 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 9. (i) Schematic representation of the multichannel device created by Kyoung Han et al. in which wax patterned-NC membranes were stacked in multiple layers (ii) to create flow paths that allow for the sequential flow of the sample and signal amplification reagents to the test line. (iii) Picture of the device after the detection of GDH and Clostridioides diff icile toxins A and B. Reprinted (adapted) with permission from ref 129. Copyright 2021 Elsevier. channel lateral flow assay for the detection of 10 foodborne friendliness of the MPQ reader, as this might hinder the pathogens (E. coli O157:H7, Salmonella paratyphi A, S. applicability of this platform in real scenarios. paratyphi B, S. paratyphi C, Salmonella typhi, S. enteritidis, 4.2. Using Nanoparticles for Multiplexing Salmonella choleraesuis, Vibrio cholera O1, V. cholera O139, and Vibrio parahaemolyticus). Each strip was functionalized with As has been seen throughout this Review so far, nanoparticles monoclonal antibodies specific to one of the pathogenic are routinely used in LFAs. By designing LFAs with different bacteria, and the conjugate pad was prepared by conjugating nanoparticles that generate different signals it is easy to the antibodies with phosphor nanoparticles. The results envisage how multiplexed assays can be developed. In such obtained were 100% consistent when validated against the systems it is important to be able to distinguish the signals culture-based detection of samples taken from 279 food quickly and easily from the different nanoparticles to allow the samples.128 Despite being fast and reliable, the use of this kind fast, efficient, and qualitative interpretation of the assay results. Recent research has also focused on the integration of low- of multiplexing format is limited by the requirement of larger cost, portable, and robust assay readout technologies. This sample volumes. In this case, the sample volume required was section of the Review will focus on novel nanoparticle LFAs 7 times higher (700 μL) than the single-strip LFAs. This that have been developed for multiplexed sample analysis. prevents the use of this multiplexing method in scenarios As well as AuNPs, latex beads are very popular nanoparticles where the sample volumes are prohibitively low. for LFA development. An example of latex beads being used in Han et al. have developed a similar strategy but, in this work, a multiplexed LFA is the work of Lee et al., who developed a stack nitrocellulose sheets on top of each other, using wax multiplex LFA that uses red and blue latex beads for the patterning to create different flow paths vertically through the detection of IgG and IgM produced as a response to infections sheets (Figure 9i). This methodology was implemented to by dengue virus (DENV) and Chikungunya virus (CHIKV). allow the sequential flow of signal amplification reagents to The appearance of a purple color on the TL indicated the increase the flow sensitivity (Figure 9ii). With this approach, presence of both IgG and IgM antibodies in the sample. A the authors achieved the simultaneous detection of Clostri- quantitative evaluation of the strips was performed with a dioides diff icile toxins A and B, and glutamate dehydrogenase smartphone camera and image analysis software. In this work (GDH) (Figure 9iii). The signal amplification strategy, based the hue (H) value was analyzed instead of the RGB value, as it on the aggregation of AuNPs, enabled the detection of the was found to be more accurate.130 Similarly, Anfossi et al. used target analytes at ultralow concentrations, with LoDs of 0.16, spherical AgNPs, spherical AuNPs, and “desert-rose-like” 0.09, and 0.03 ng mL−1 for GDH, C. dif f icile toxin A, and C. AuNPs, with SPR peaks at 420, 525, and 620 nm, respectively, diff icile toxin B, respectively.129 to develop a multiplexed LFA for detection of casein, A multiplexing strategy based on the interrogation of three ovalbumin, and hazelnut allergenic proteins in commercial individual LFA strips using a single processor unit has been biscuits. The LFA comprises three lines, each responsive to reported by Orlov et al. In this work magnetic nanoparticles one allergen. In order to obtain equivalent signal intensities were conjugated to monoclonal antibodies and used as labels, from the different nanoparticles, the authors had to increase with each strip responsible for the detection of a particular the concentration of AgNPs in the conjugate pad. This is not target analyte. The assays were developed to detect Botulinum surprising since AgNPs have lower extinction coefficients than neurotoxins (BoNTs) A, B, and E in whole milk and juice AuNPs. The results of this particular multiplexed assay are easy samples and were simultaneously analyzed using a magnetic to interpret since the colors of each nanoparticle (cyan, yellow, particle quantification (MPQ) reader.56 The use of this type of and magenta) can be easily interpreted using conventional signal transduction enabled high assay sensitivity compared to image analysis software.131 the conventional colorimetric readout, allowing the detection The appropriateness of the colorimetric LFA is usually of the target analytes in the low pg mL−1 range. The authors diminished by the low sensitivity and ease of quantification of observe a linear range across 7 decades in this work which this type of signal readout. Quantum dots (QDs) have highlights the suitability of this method for multiplexed LFAs advanced optical properties that make them ideal for the where analytes can span wide concentration ranges. Special development of highly sensitive, easily quantifiable multiplexed attention should be given to the price, portability, and user- LFAs. However, the fluorescence emission of QDs can be 14896 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 10. (A, i) SEM and TEM images of the ZrMOF@CdTe NPs, which show a cubic shape and a size around 120 nm. (A, ii) Multiplexed LFA based on the use of two TLs and ZrMOF@CdTe NPs with yellow and green fluorescent emissions. Reprinted (adapted) with permission from ref 133. Copyright 2021 The Royal Society of Chemistry. (B, i) Multiplexed LFAs showing the simultaneous detection of the staphylococcal enterotoxins I, G, and H, using lanthanide-doped nanoparticles. (B, ii) Smartphone-based readout system for the on-site LFA’s quantification. Reprinted (adapted) with permission from ref 134. Copyright 2021 The Royal Society of Chemistry. suddenly quenched by biomolecules present in biological immobilization on three consecutive TLs in the detection pad samples, compromising the sensitivity and reliability of the (Figure 10Bi). This multiplexed assay was 100 times more assay.132 To resolve this, recent research has investigated the sensitive than the comparative AuNP-based LFA, reaching encapsulation of QDs into nanoparticles that shield them from LoDs in the pg mL−1 range for all the biomarkers investigated. quenchers. For instance, Zou et al. have reported the Additionally, the researchers developed a portable and cost- encapsulation of CdTe QDs into cubic zirconium metal effective smartphone-based readout system (see section 2; organic frameworks (ZrMOFs; approximately 120 nm long) Figure 10Bii).134 (Figure 10Ai), and their use as fluorescent labels for the 4.2.1. Multiplexing in a Single TL. The detection of simultaneous detection of heart-type fatty acid binding protein different target analytes within a single TL is probably the most (h-FABP) and cardiac troponin T (cTnT). This nanomaterial powerful method of multiplexing in LFAs. This retains the is synthesized with a simple hydrothermal method, in which simplicity of a conventional double-line LFA, in terms of both the reaction time can be modulated to obtain ZrMOF@CdTe fabrication and readout of results. Again, multiplexing in one NPs with different fluorescent properties. The authors have TL can be coupled to printing several TLs to exponentially taken advantage of this to develop a multiplexed LFA that uses increase the multiplexing capability of an LFA. To perform two TLs for ZrMOF@CdTe NPs that emit yellow and green multiplexed detection in a single TL, several labels with easily light after being excited by a single wavelength (Figure 10Aii). differentiated signals must be used. For instance, Yen et al. This approach yielded visual LoDs of 1 and 250 μg L−1 for h- utilized the size-dependent optical properties of AgNPs to FABP and cTnT, respectively, below the lowest clinically develop a multiplexed LFA for the detection of the dengue relevant concentration. In addition, the researchers report a virus (DENV) NS1 protein, the yellow fever virus (YFV) NS1 high specificity over other cardiac biomarkers [including D- protein, and an Ebola virus (ZEBOV) glycoprotein. The three dimer (D-D), myoglobin, and C-reactive protein (CRP)].133 monoclonal capture antibodies were deposited in equal Lanthanide-doped nanoparticles (YVO4/Eu 40%) have been concentrations on a single TL, and the conjugated pad was shown by Mosseau et al. to possess remarkable optical loaded with a mixture of orange, red, and green AgNPs, each properties compared to the QDs, due to an absence of functionalized for a separate target analyte. The presence of the photobleaching and longer luminescence times. One of the three proteins in the sample turned the TL brown. The assay advantages of this work is the ease of nanoparticle synthesis was shown to be quantifiable using RGB analysis with a which is achieved by the coprecipitation YVO4 and Eu salts. smartphone camera.135 Similarly, Zhang et al. have developed a These nanoparticles were used for the simultaneous detection multiplexing strategy for the detection of three cardiac disease of staphylococcal enterotoxins G, H, and I, by the conjugation biomarkers for the early detection of acute myocardial of the nanoparticles with monoclonal antibodies and the infarctions [creatine kinase-MB isoenzymes (CK-MB), cardiac 14897 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 11. Multiplexing strategy based on the detection of CK-MB, cTnI, and myoglobin cardiac biomarkers in a single TL, using three Raman reporters. Reprinted (adapted) with permission from ref 136. Copyright 2018 Elsevier troponin I (cTnI), and myoglobin] in a single TL, using SERS conditions can even clog the nitrocellulose pores and prevent signal transduction (Figure 11). In this work antibody- the sample from reaching the detection zone.81 Likewise the functionalized silver core and gold shell NPs loaded with opacity of certain real samples such as whole blood16 can mask three different Raman dyes [methylene blue (MB), Nile blue A the optical signal generated at the TL. Common methods to (NBA), and rhodamine 6G (R6 G)] were used (Figure 11). overcome this include the incorporation of sample dilution or The authors reported ultralow detection limits (in the low pg washing steps,155 often including anionic surfactants such as mL−1 range) and wide linear dynamic ranges (3 orders of Tween-20156 and sodium dodecyl sulfate.157 magnitude) that cover the clinical ranges of the three The analytical sensitivity and specificity of LFA are biomarkers. This multiplexing approach is particularly useful frequently impaired when evaluating real samples due to the for SERS, since Raman mapping can be time-consuming. In presence of analytes that are similar to the biomarker of this work the time to result was reduced from 21 min for three interest. It is therefore vital to identify a bioreceptor that is consecutive TLs to 7 min for a single multiplexed test line.136 highly specific to the biomarker of interest.158 Any assay is only going to be as good as the bioreceptor it uses. Assay 5. EVALUATION OF COMPLEX SAMPLES performance can be improved by the incorporation of sample pretreatment methods such as analyte preconcentration (e.g., One of the biggest challenges in LFA development is to magnetic pre-enrichment159 and nucleic acid amplifica- achieve high analytical capabilities in complex real-world tion151,160) or filtering.161 However, these usually increase samples. This is a fundamental issue that must be resolved the number of steps, reagents, ease-of-use, and time to result of to upgrade the technology readiness level (TRL) to more than the assay. 4 (see section 6).137 Most real-world samples contain a It is important to optimize all these conditions, especially multitude of components that can rapidly and drastically vary when trying to improve the sensitivity of LFAs for the analysis in composition; these variations alter the physicochemical of low-concentration biomarkers such as tau proteins properties of the assay.138 For example, the pH and ionic (Alzheimer disease),162 soluble CD25 (hepatocellular carcino- strength of the sample can affect the stability and binding ma),163 and tumor necrosis factor-α (obstructive sleep apnea constant of the bioreceptors to the target analyte.139 Therefore, syndrome)164 that are present at trace levels (pg mL−1 range). any fluctuations of these in the sample can vastly affect the In this section of the Review, we will discuss recent analyte capture and therefore assay quantification. advancements and novel methodologies to better the analytical The optimal working conditions for most antibodies are at performance of LFAs in complex media. pH 7.415,140,141 and ionic strengths of around 30 mM (although low ionic strength might also contribute to 5.1. Sample Filtration nonspecific protein−protein interactions).142,143 Nucleic-acid- One of the most straightforward ways to purify the sample and based probes have an optimal working pH of 7.5−8.5144 and preconcentrate the target analyte is by using external or show better affinity at higher salt concentrations (>100 integrated filtration membranes. At the time of writing, 0.45 mM).145−151 It is important to know the pH and ionic μm pore diameter cellulose nitrate filters are routinely used for strength of the media in which the LFA will be run as these can the preconcentration of bacteria in water samples.165 vary drastically, not just between media but also in a single Integrated filters are also commonly used in LFAs that analyze media over time. For instance, saliva has a pH of 7.3−7.4 and whole blood samples, primarily for the removal of red blood ionic strength of 0.15 M,152 whereas sweat has a much lower cells. Two commercially available plasma separation mem- pH of between 4 and 6 and an ionic strength that varies greatly branes are Cytosep and Vivid plasma;161,166 examples for their with sweat rate, location on the body, and evaporation.153,154 use in LFAs include the detection of the capsid protein VP72 One method to overcome this is to adjust the pH and ionic from the African swine fever virus167 and the diagnosis of strength of the samples to the optimal assay conditions before SARS-CoV-2168 and antihuman immunodeficiency virus performing the assay. This is done by drying a buffer into the (HIV).169 sample pad of the LFA. There are physical as well as chemical One of the drawbacks to these types of filters is that the properties of the sample that can affect the LFA; for example, small pore sizes of the membranes (<9 μm) can promote the sample viscosity will affect the flow rate and in extreme blood clotting (Figure 12Ai), which limits the plasma/serum 14898 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 12. (A, i) Schematic representation of red blood cells clogging the filtration membrane. (A, ii) The use of the differential pad avoids the formation of the blood fouling layer, while the filtration and calibration pads are responsible for the separation and storage of the serum sample, respectively. Reprinted (adapted) with permission from ref 170. Copyright 2018 The Royal Society of Chemistry. (B, i) Schematic representation of the integrated PCR amplicon purification approach based on the immobilization of functionalized graphene oxide on the sample pad. (B, ii) The three-layered GO sample pad enables the scrubbing of almost all the residual primers and primer-dimers with <40 pb out of the sample matrix. Reprinted (adapted) with permission from ref 171. Copyright 2017 American Chemical Society. Figure 13. (A) Schematic representation of a 10 min preconcentration approach based on the incorporation of a dialysis membrane onto the sample pad of an LFA strip. PEG conjugated to a fiberglass membrane is used as the dialysate. Reprinted (adapted) with permission from ref 172. Copyright 2016 Elsevier. (B) Schematic representation of a paper electrophoretic device powered by a smartphone battery. The whole blood sample is driven by electrophoresis toward the detection zone, avoiding the clogging on the nitrocellulose pores. Reprinted (adapted) with permission from ref 71. Copyright 2021 American Chemical Society. 14899 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review separation yield to just 10−30%.170 To this end, Lu et al. have A potential of 200 V was applied across the nitrocellulose test developed a low-cost (<$0.10) cross-flow filtration pad that strip using a smartphone in combination with a joule thief increases the separation yield by up to 86%. This method inverter and rectifier, which is compatible with the use of this requires three paper-based membranes stacked on top of each assay in PoC scenarios where resources are limited. The other. The top “differentiation pad” (40 μm pore size) is electrophoresis drives the whole blood sample to the detection responsible for preventing the formation of the blood fouling zone, avoiding the clogging of the nitrocellulose pores. Any layer. The middle “filtration pad” (1.9 μm pore size) is excess blood can then be electrophoretically driven off of the responsible for filtering out the red blood cells, and the final detection zone allowing a clear visual readout that can be easily “calibration” membrane (also 1.9 μm pore size) is used to store quantified using a smartphone for image capture and analysis the sample (liquid holding capacity of 40 μL cm−2; Figure (Figure 13B). This approach allowed for the assay sensitivity in 12Aii). The authors have applied this approach for the whole blood to reach the same levels of those obtained in the multiplexed detection of CRP, RBP, and ferritin, achieving lab with PBS. These methodologies are new and require similar analytical accuracy as standard centrifuge-based serum further optimization and analysis but clearly have the potential separation methods.170 for use in future LFAs in complex media. In certain situations, it is known what molecules will be 5.3. Bioreceptor Engineering present that need to be removed in order to improve the assay performance. In such situations, the filters can be function- A more sophisticated way to improve the analytical capability alized to specifically remove known contaminants. One such of LFA when evaluating complex samples is to improve the example of sample scrubbing has been shown by Li et al., who analytical properties of the bioreceptors. Traditional bio- have reported an innovative strategy to purify PCR amplicons receptors include DNA fragments and antibodies, but in recent directly in the LFA strip. They integrate high-surface-area years, novel bioreceptors such as aptamers and antibody graphene oxide (GO) functionalized for the specific capture of fragments have garnered much interest. their PCR primers into the sample pad of their LFA (Figure Aptamers are nucleic acid sequences (normally DNA or 12Bi). They report the adsorption of almost all the residual RNA) that fold into three-dimensional conformations that primers, and primer-dimers with less than 40 bp, within the allow them to specifically bind to analytes of interest.175 sample pad, while their 106 bp amplicons were able to flow to Aptamer selection is performed through a controlled in vitro the test line (Figure 12Bii). With this approach, the authors process (using the Sequential Evolution of Ligands by achieved a 1000-fold sensitivity enhancement for the detection Exponential Enrichment, SELEX) that allows not only for of bacteriophage λ-DNA, obtaining an LoD of 30 copies by nonselective sequences to be discarded but also for those naked eye.171 sequences that do bind to the analyte of interest to be ranked and selected based on their binding affinity.176 While the 5.2. Sample Pretreatments Based on Active yet Automatic binding constants tend to be similar to those of antibodies, Manipulation of the Sample aptamers generally produce a more specific response. There are Sample preconcentration can also be performed by the three main reasons for this: (i) They have lower molecular integration of physical separation techniques in LFA. For weights than antibodies, decreasing the chances of nonspecific example, Tang et al. have integrated dialysis into an LFA for interactions. (ii) Being nucleic acids, their interaction with the preconcentration of the sample. They incorporate a other bioreceptors is lower. (iii) During the SELEX process, it semipermeable membrane (MWCO 3.5 KD) onto a fiberglass is possible to include a counter-selection step, which actively sample pad that has been pretreated with PEG. The highly removes all aptamer sequences that nonspecifically bind to hygroscopic nature of the PEGylated fiberglass allows it to act possible contaminants. Huan et al. have recently reviewed the as a dialysate in this system (Figure 13A). With this approach, application of aptamers in LFAs, focusing on the combination the authors report that a 10 min preconcentration resulted in a of these bioreceptors with signal amplification and multiplexing 10-fold and 4-fold signal enhancement in the detection of HIV strategies.177 specific nucleic acid sequences and myoglobin, respectively. Similar to aptamers, antibody fragments and nanobodies can This strategy shows great promise for PoC applications given also improve the specificity of LFA when employing complex its simplicity, speed, and low cost; however, the approach still samples. Antibody fragments are generated by the proteolytic needs to be validated with real-world samples.172 enzymatic fragmentation of full size antibodies (mostly IgG). Moghadam et al. have used isotachophoresis in LFAs for In this way it is possible to remove the Fc region of IgG, which integrated sample extraction and preconcentration. Isotacho- is usually responsible of nonspecific interactions with phoresis is a nonlinear, equipment-free, electrophoretic endogenous Fc receptors. Additionally, the presence of less technique where leading (LE) and trailing (TE) electrolytes charged groups reduces the possibility of cross-reactivity.19 are used to focus the analyte in a reduced area. This technique However, to the best of our knowledge there are still no was found to improve the binding affinity between human IgG reports proving the aforementioned analytical capabilities of (HIgG) and anti-HIgG resulting in a 1 order of magnitude antibody fragments when evaluating complex samples in LFA. increase in the concentration of analyte bound at the TL and a On the contrary, nanobodies (or single domain antibodies, 400-fold improvement in the LoD of the assay, when sdAb) are the single chain binding portions of camelid compared to the conventional LFA. Additionally, the authors antibodies. Thanks to their low molecular weight (<15 kDa), show that isotachophoresis improved analyte extraction from easy fabrication (being a single chain of amino acid, they can the sample matrix; in their case, up to 80% of IgG could be be produced by E. coli in vitro), and stability, they seem like an extracted from real human serum samples.173,174 ideal bioreceptor for LFA. In this context, few works have On a similar note, Sena et al. have developed a portable and employed them to enhance the LFA capabilities. Between low-cost paper-based electrophoretic bioassay (PEB) that them, the work from Loynachan et al. combined nanobodies enables the detection of HIgG in neat whole blood samples. with platinum nanocatalysts to achieve the naked-eye detection 14900 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review Figure 14. (A) Schematic representation of the face mask-LFA capable of detecting exhaled SARS-CoV-2. Once the water reservoir is pressed, the water flow moves the collected aerosol sample toward the paper μPAD, which contains the freeze-dried assay reagents. (B) First, the RNA is extracted, and then, it is amplified by RT-RPA and detected by the Cas12a SHERLOCK method. The output is visualized in an LFA strip that is built into the face mask. (C) Pictures of the face mask interior and exterior. Reprinted (adapted) with permission from ref 181. Copyright 2021 Springer Nature. of p24 spiked into sera in the low femtomolar range, as well as As with any technology, one method of estimating how close the detection of acute-phase HIV in clinical human plasma a particular LFA is to market is by assessing its TRL level. This samples.178 is a system used to assess the maturity of a particular The implementation of the CRISPR/Cas recognition technology.137 For the different stages of LFA development, systems in LFAs has been reported to provide both high 1. TRL-1 would correspond to the achievement of a specificity and sensitivity.179 This method has recently been measurable signal in the TL upon the detection of a utilized for the dual detection of the envelope (E) and open target analyte using a half-stick format; reading frame 1ab (Orf1ab) genes of SARS-CoV-2 in nasopharyngeal swab samples by Xiong et al. The use of 2. TRL-2 to the identification of the ideal components to build the full LFA; CRISPR/Cas9 with multiplexed RT-RPA enabled the amplification of the target genes without the need for external 3. TRL-3 to the determination of the LFA properties instrumentation. Cas9/sgRNA complex formation eliminates (sensitivity, specificity, dynamic range, RSD, etc.) after interference from the residual primer-dimers, improving the the evaluation of serial dilutions of the analyte in buffer assay specificity. The authors report a high clinical sensitivity in the laboratory; (97%) and specificity (100%) after evaluating 64 real patient 4. TRL-4 to the determination of the LFA accuracy when samples.180 detecting the analyte in real samples (prevalidated using Nguyen et al. have developed an interesting platform that gold standard methods) in the laboratory; utilizes isothermal amplification and Cas12a SHERLOCK for 5. TRL-5 to the validation of the LFA with prevalidated the detection of exhaled SARS-COV-2 on a face mask. The real samples in the hospital with trained users; aerosol collection and nucleic acid amplification are performed 6. TRL-6 to the validation of the LFA with clinical samples on the interior surface of the face mask, while the detection in the hospital with trained personnel; step is done by the integration of a dedicated LFA (Figure 14). 7. TRL-7 to the validation of the LFA in the hospital using This approach showed high selectivity over other human clinical samples and untrained personnel; coronavirus strains and had an LoD of 500 RNA copies.181 8. TRL-8 to the assessment of the commercial LFA in deployment conditions, proving full compliance of the quality standards and regulations; and 6. LFA VALIDATION AND BARRIERS TOWARD 9. TRL-9 to the achievement of a fully operational LFA, COMMERCIALIZATION ready for commercialization, with the system optimized The LFA market has seen a fascinating growth in recent for full rate production. decades, from USD 2.2 billion in 2005 to USD 8.2 billion in Most of the technology (with a few exceptions) discussed in 2020, which has been driven by the high prevalence of sections 2−5 of this Review are at TRL levels 1−5. infectious diseases and the increasing popularity of home- Commercially available LFAs are relatively simple devices diagnosis.182 The outbreak of the COVID-19 pandemic caused not utilizing the technological advancements being reported in a boom in the market by rapidly increasing the demand for at- research laboratories. This begs the question of what barriers home (PoC) test kits.183 Despite their lower accuracy are preventing the commercial adoption of these technological compared to RT-qPCR, LFAs have still proven popular due advances? to their point-of-care properties. Given this, the LFA market One possible reason is that research centers and small size is expected to reach USD 10.2 billion by 2025.184 companies suffer a funding gap between TRLs 4 and 6, also 14901 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review known as the “valley of death”.185 This is the point in assay enhancement, multiplexing, quantification at the PoC, and development when tests move away from in-the-lab measure- evaluation of complex samples. These improvements need to ments with ideal conditions and simulated samples (TRL 4) to retain the key advantageous properties of LFAs, namely, their validation with real-world samples at the intended point of use low cost, rapid time to result, and ease of operation (TRLs 5 and 6). Most investors only become interested when interpretation. technologies reach TRLs 7−9.186 In an attempt to correct this Notable advances have been made that increase the issue, funding organizations (e.g., the European Commission) sensitivity of LFAs. These are important as assays are being and governments have started to increase funding to developed for low-concentration analytes, increasing the utility collaborative projects between academia and industry.187−189 of LFAs. Sensitivity can be enhanced using novel reporters, The involvement of industrial partners throughout the entire signal amplifiers, and alternative signal transduction methods. LFA development process is intended to encourage stricter Likewise, advances in multiplexing methodologies have been critical evaluation of LFA cost, performance, manufacturing reviewed and are sure to form a large part of future research capability, and scalability, properties that are often overlooked and commercial LFA products. Technologies that allow the in academic research. In addition to this, the involvement of detection of multiple analytes in a single TL are perhaps the industrial partners as early as possible is intended to speed up most interesting as they allow the exponential increase in the the time to market of LFAs.190−192 number of analytes that can be detected in an LFA, since they Another important barrier toward commercialization is to can be combined with novel device designs that allow multiple obtain all the regulatory approval required for product launch. target analytes to be investigated in a single test. Likewise, This is a tedious and expensive process that starts with the microarrays optimize the available test area for the detection of analytical and clinical validation of the test. The analytical a multitude of analytes in a single detection zone. validation assesses the precision, accuracy, and reliability of the While there has been a miniaturization of LFA readers in test, while the clinical validation evaluates the parameters such recent years to analyze and quantify the signal on test strips, as the diagnostic sensitivity, specificity, positive and negative the past decade has seen a real trend in the use of smartphones predictive value, and likelihood ratio of the LFA.193 There are for LFA analysis. This often requires portable boxes or several companies offering support services to diagnostic cassettes to control the lighting conditions of image acquisition companies during this process.194,195 with a smartphone camera. The advent of portable 3D printers For goods intended to be sold in the European Union (EU), is allowing the fabrication of many of these at the point of use. a CE mark is required. In order to obtain this certification, LFA In the future the increasing power of smartphones and the manufacturers must submit a performance evaluation report to advent of 5G networking promise to further improve PoC the regulatory agency showing the analytical and clinical LFAs, in terms of both their analytical performance and their validation of their LFA. The in vitro diagnostic medical devices data analysis. The development of point-of-care readers that regulation (IVDR) (EU, 2017/746) classifies devices in enable PoC analysis using alternative detection methods (such relation to their risk if inaccurate; in other words, if the test as SERS, TCA, and MPQ) is still in its infancy but gives a false negative, what risk is posed to the general undoubtedly has potential. We anticipate that the next decade population. Tests are assigned a letter of the alphabet (A, B, C, will see an increase in research to reduce the cost and improve or D) based on their score in this test. Diagnostics for the the performance of such readers for LFA applications. detection of pregnancy, cholesterol level, glucose, or bacteria in The simplicity of use of LFAs is what makes them so urine are classified as moderate health risks (B), while assays desirable; it is therefore encouraging to see the work by for transmissible agents with a high risk of propagation (e.g., researchers to incorporate sample pretreatment methods into SARS-CoV-2) are classified as D. The next step is a conformity LFAs. Any technological developments aimed at allowing the assessment, in which an independent certified body evaluates analysis of untreated sample matrices is of great interest to the the analytical and clinical performance of the test.196 entire field of LFA development. The future of LFAs is In America, the Food and Drug Administration (FDA) has a undoubtedly a “closed box” system for the end user where they similar regulation, where the tests are classified as I, II, or III add their sample and get a simple, reliable, and quantified depending on their health risk in case of inaccuracy. Most readout. Such devices will most likely include combinations of LFAs are class II and must undergo a stringent premarket the technologies reviewed herein. The practice of combining review (only class I is exempt), where their analytical and technologies is rare, with most publications understandably clinical performance is evaluated and compared with focusing on a single, novel technological advancement. The equivalent tests or with a standardized reference method.197 combination of several technological advancements in a single The European and American regulators are the strictest and LFA has the potential to make a greater societal impact and hardest to obtain. If a test is developed that passes these should be given encouragement. regulations, then they will normally be accepted worldwide. Throughout this Review, we have seen that there are various The main obstacles to commercialization are therefore not techniques that have been employed to address the four main necessarily scientific issues, but rather difficulties in raising challenges in the field: quantification at the PoC, sensitivity capital for low TRL level technologies and the high costs (both enhancement, multiplexing, and the evaluation of complex financial and in terms of time) and complexity of obtaining samples. While all of the techniques investigated require more regulatory approval. development, it is worth considering which are currently the most promising. This is a largely subjective exercise, but the 7. OUTLOOK authors would suggest that, of all the techniques covered, In this paper, we have reviewed the advances in LFA from the perhaps the use of SERS in LFAs is the most advanced and last ten years with an emphasis on the role of nanomaterials. currently the best able to solve the four main challenges We have identified four main areas that should simultaneously highlighted in this Review. We have seen that portable SERS be improved to achieve the next generation of LFA: sensitivity readers are being successfully developed for on-site quantifi- 14902 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review cation of signals in LFAs;59 there are improved sensitivities Politècnica de València, Universitat de València, 46022 when using SERS compared to colorimetric detection, with Valencia, Spain LoDs as low as pg/mL being reported.123 Different SERS Ruslan Á lvarez-Diduk − Nanobioelectronics & Biosensors nanotags that emit different SERS signals have been employed Group, Institut Català de Nanociència I Nanotecnologia for multiplexing,136 and while SERS is naturally more suited to (ICN2), CSIC and The Barcelona Institute of Science and complex media analysis than colorimetric or fluorescent Technology (BIST), 08193 Barcelona, Spain; orcid.org/ measurements, the ability to couple SERS labels to magnetic 0000-0002-9876-1574 nanoparticles has also facilitated complex media analysis.103 Claudio Parolo − Barcelona Institute for Global Health The greatest challenges in SERS-based LFA development (ISGlobal) Hospital Clínic−Universitat de Barcelona, 08036 concern the readers, which need much more development to Barcelona, Spain; orcid.org/0000-0001-9481-4408 be practically usable. Current limitations include high costs, Andrew Piper − Nanobioelectronics & Biosensors Group, long data acquisition times, and not being very user-friendly. Institut Català de Nanociència I Nanotecnologia (ICN2), The authors believe work should be conducted to increase the CSIC and The Barcelona Institute of Science and Technology scan speed and software developed to give simple-to- (BIST), 08193 Barcelona, Spain understand readouts. Unlike optical readout methods, the Complete contact information is available at: results of SERS are not intuitively understood, so software that https://pubs.acs.org/10.1021/acs.chemrev.1c01012 can accurately and reliably process, interpret, and report the results in an easy-to-understand fashion is required for real- Notes world applications where the assays will be performed by untrained personnel. The authors declare no competing financial interest. Advances in the other technologies discussed in this Review Biographies (such as TCA) show that they are very promising and developing well; however, at the time of writing, they are not Dr. Amadeo Sena-Torralba holds a M.Sc. and Ph.D. in advanced able to solve all four of the issues discussed (multiplexing with biotechnology from the Autonomous University of Barcelona, under TCA has not been demonstrated yet). Further development of the supervision of Prof. Arben Merkoçi [Catalan Institute of these techniques to address these issues is required, before Nanoscience and Nanotechnology (ICN2)]. His doctoral research integrating those developments into a single sensing model, was focused on the development of innovative paper-based nano- which means they are some years behind SERS. Similarly, the biosensing platforms, with applicability at the point-of-care. He has recently published work by Miller et al. also achieves received the Elvira Rodenas Ciller 2021 award, conferred by the GECI quantification and ultralow sensitivity;84 however, it would group of the Spanish Royal Society of Chemistry (RSEQ) and Physics probably require extensive sample treatment, and its ability to (RSEF) to the best doctoral thesis on colloids and interfaces sciences. detect multiple targets would require the printing of multiple He is currently working as an awardee of the Spanish Juan de la detection zones. Cierva postdoctoral fellowship, at the Instituto Interuniversitario de There is an obvious trend in the literature to develop new Investigación en Reconocimiento Molecular y Desarrollo Tecnológi- methods of signal transduction. There have been decades of co. research and development of optical (colorimetric and Dr. Ruslan Á lvarez Diduk obtained his Ph.D. in chemistry at the fluorescent) LFAs, and their limitations seem to have been Universidad Autónoma Metropolitana (México) in 2014. From 2015 reached. They lack the sensitivity and reliability to detect the to date, he works as a postdoctoral researcher in the Nano- biomarkers of interest for future sensing applications, currently bioelectronics and Biosensors group (Prof. Merkoçi). His research only being able to detect highly concentrated biomarkers. focuses on the development of novel nanostructured biosensors using Different types of energy (sound and thermal) and forces both optical and electrochemical platforms, which are based on point- (magnetism) are being used to replace light in LFAs; we of-care and low-cost systems. He is also involved in exploring new and envisage that in the future more development of these and the novel routes for nanoparticle synthesis, graphene reduction and laser use of other signal transduction methods will be prominent in patterning, and the development of readout platforms including LFA development. smartphone-based biosensing. AUTHOR INFORMATION Dr. Claudio Parolo is currently a senior scientist at the Barcelona Corresponding Author Institute for Global Health. After obtaining his Ph.D. from the Autonomous University of Barcelona under the supervision of Prof. Arben Merkoçi − Nanobioelectronics & Biosensors Group, Merkoçi, he worked as postdoctoral fellow on some of the world- Institut Català de Nanociència I Nanotecnologia (ICN2), leading laboratories in the field of diagnostics, including Whitesides’, CSIC and The Barcelona Institute of Science and Technology McKendry’s, and Plaxco’s groups. In February 2021, he joined (BIST), 08193 Barcelona, Spain; Catalan Institution for ISGlobal, and the team working on tropical and neglected diseases. Research and Advanced Studies (ICREA), 08010 Barcelona, Specifically, he is currently developing lateral flow assays and Spain; orcid.org/0000-0003-2486-8085; electrochemical diagnostic devices for the detection at the point of Email: arben.merkoci@icn2.cat care of malaria and dengue biomarkers. Authors Dr. Andrew Piper obtained an undergraduate taught Masters degree Amadeo Sena-Torralba − Nanobioelectronics & Biosensors in Medicinal and Biological Chemistry from the University of Group, Institut Català de Nanociència I Nanotecnologia Edinburgh. He stayed at the University of Edinburgh to obtain a (ICN2), CSIC and The Barcelona Institute of Science and Ph.D. in nanoscale biosensor development under the supervision of Technology (BIST), 08193 Barcelona, Spain; Instituto Prof. Andrew R Mount. He has held postdoctoral research positions Interuniversitario de Investigación de Reconocimiento at the University of Oxford as well as the Royal Institute of Molecular y Desarrollo Tecnológico (IDM), Universitat Technology (KTH) and the Karolinska Institutet in Stockholm, all on 14903 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. Rev. 2022, 122, 14881−14910 Chemical Reviews pubs.acs.org/CR Review the development of biosensors/diagnostics. He is currently based at CFU = colony-forming unit the Catalan Institute of Nanoscience and Nanotechnology (ICN2) as CK-MB = creatine kinase myocardial band part of the Merkoçi research group where he overseas projects CL = control line focussing on several different types of nano-biosensor development. CMOS = complementary metal-oxide-semiconductor Prof. Arben Merkoçi is an ICREA Research Professor and leader of CPU = central processing unit the ICN2 Nanobioelectronics and Biosensors Group. He is also the CrAg = cryptococcal antigen Co-Editor in Chief of the Biosensors and Bioelectronics journal and CRISPR = clustered regularly interspaced short palindromic member of the editorial board of Electroanalysis, Microchimica Acta. repeats Prof. Merkoçi has published 319 articles (H-index/citations: Google cTnI = cardiac troponin I Scholar 87/25890; WOS 70/17682) and supervised 35 Ph.D. theses. C. diff icile = Clostridium dif ficile He serves also as a scientific evaluator and member of panels of DENV = dengue virus experts of various international governmental and nongovernmental DNA = deoxyribonucleic acid agencies (EU-FP and EU-ERC panels and other panels in Europe, DTNB = 5,5′-dithiobis(2-nitrobenzoic acid) USA, and other countries) and is the cofounder of two spin-off E. coli = Escherichia coli companies: GraphenicaLab, devoted to graphene patterning, and ELISA = enzyme-linked immunosorbent assay Paperdrop Diagnostics, dedicated to clinical diagnostics. Ems. = emission et al. = et alia (and others) Exc = excitation ACKNOWLEDGMENTS EVD = Ebola virus disease We acknowledge financial support to the project AC21_2/ FAM = carboxyfluorescein 00044 (GLEBIOASSAY), funded by Instituto de Salud Carlos FDA = Food and Drug Administration III (ISCIII) and cofunded by the European Union, MICROB- GDH = glutamate dehydrogenase PREDICT project (European Union Horizon 2020 research GO = graphene oxide and innovation program under grant agreement 825694), and GPS = global positioning system Graphene Flagship Core 3 (European Union Horizon 2020 HAdV = human adenovirus research and innovation program under grant agreement HAV = hepatitis A virus 881603). ICN2 is funded by CERCA programme, Generalitat hCG = human chorionic gonadotropin de Catalunya. Grant SEV-2017-0706 is funded by MCIN/ HCV = hepatitis C virus AEI/10.13039/501100011033. The authors also acknowledge HIgG = human immunoglobulin G the Project PID2021-124795NB-I00 funded by MCIN/AEI/ HIV = human immunodeficiency virus 10.13039/501100011033/and FEDER Una manera de hacer HPV = human papillomavirus Europa and the project PAPYRUS: Grant PLEC2021-007972 HRP = horseradish peroxidase funded by MCIN/AEI/10.13039/501100011033 and by the H1N1 = subtype of the type A influenza virus “European Union NextGenerationEU/PRTR”. The authors IgG = immunoglobulin G acknowledge Consejo Superior de Investigaciones Científicas IgM = immunoglobulin M (CSIC) for the project “COVID19-122” granted in the call ITP = isotachophoresis “Nuevas ayudas extraordinarias a proyectos de investigación en IU = international units el marco de las medidas urgentes extraordinarias para hacer IVD = in vitro diagnostics frente al impacto económico y social del COVID-19” (Ayudas IVDR = in vitro diagnostics regulations CSIC-COVID-19). A.S.-T. acknowledges MINECO for the KD = thermodynamic equilibrium dissociation constant Juan de la Cierva Formación fellowship (FJC2020-043927-I). kDa = kilo-Dalton C.P. acknowledges the Marie Skłodowska-Curie Actions LATE = linear-after-the-exponential Individual Fellowship; this project received funding from the LDW = laser direct writing European Union’s Horizon 2020 research and innovation LE = leading electrolyte programme under the Marie Skłodowska-Curie grant agree- LED = light-emitting diode ment 795635. C.P. (ISGlobal) also acknowledges support from LFA = lateral flow assay the Spanish Ministry of Science and Innovation** and State LoD = limit of detection Research Agency through the “Centro de Excelencia Severo LoQ = limit of quantification Ochoa 2019−2023” Program (CEX2018-000806-S). LSPR = localized surface plasmon resonance Meth = methamphetamine LIST OF ABBREVIATIONS miRNA = micro-RNA AgNBA@Au = silver core and gold shell NPs loaded with MPQ = magnetic particle quantification Nile blue A dye MWCNTs = multiwalled carbon nanotubes AgNPs = silver nanoparticles MWCO = molecular weight cut-off AFB1 = aflatoxin B1 Myo = myoglobin ALP = alkaline phosphatase NIR = near-infrared radiation AuNPs = gold nanoparticles NPs = nanoparticles BoNT = botulinum neurotoxin N,S-doped = nitrogen- and sulfur-doped CAS = cellular apoptosis susceptibility Orf1ab = open reading frame 1ab CCD = charge-coupled device PA = photoacoustic CdSe@ZnS QDs = cadmium selenide/zinc sulfide quantum PBS = phosphate buffer saline dots PCL = polycaprolactone CdTe QDs = cadmium telluride quantum dots PCR = polymerase chain reaction 14904 https://doi.org/10.1021/acs.chemrev.1c01012 Chem. 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