Bottom‐up DNA nanostructure‐based paper as point‐of‐care diagnostic: From method to device

Paper‐based devices have attracted considerable attention in point‐of‐care testing (POCT) due to their simple operation and portability. The performance of paper‐based POC assays is further enhanced by coupling with functional nucleic acids (FNAs), which are able to selectively recognize and bind to targets, amplify and transduce signals. Several high‐performance paper‐based POC assays have been developed, resulting from the different spatial structures of the paper‐based device and detection strategies using different FNAs. In this review, we first introduce FNAs and paper‐based devices, including their concepts, classifications and advances. The following section focuses on the application of these FNAs in POC assays using paper‐based devices, taking into account their spatial one‐, two‐ and three‐dimensional structures. Finally, the challenges and perspectives of the application of FNAs in paper‐based POCT are discussed.

the practical implementation and research of paper-based POC assays.
6][17][18][19][20] FNAs have been used extensively as a tool for target recognition and signal amplification, with the specificity to detect single nucleotide polymorphisms (SNPs), 21 sensitivity at the aM level 22,23 and the potential for multi-throughput detection. 246][27] In addition, a review outlines advances in FNA-based paper devices, including DNA aptamer-and DNAzyme-coupled lateral flow assays (LFAs) and microfluidic paper-based analytical devices (μPADs), as well as modalities for signal readout. 28However, none of these reviews comprehensively cover FNAs coupled paper-based systems, nor do they discuss examples from the perspective of their relevance to POC diagnostics, which is the focus of this review.
In this review, we will focus exclusively on ultrasensitive paper-based POC assay platforms employing FNAs.Starting with the conceptual connotations of FNA, we describe the various aspects of paper-based devices coupled with FNAs, including their types, findings, structures, functions and applications.We also describe how the FNAs can be utilized for target recognition, signal amplification and coupling to paper-based devices.Afterward, the development, construction and application of paper-based devices are discussed.Particular emphasis is placed on the one-, two-and three-dimensional perspectives of POC assay strategies employing FNAs and paperbased devices.

| PAPER-BASED DEVICES
Paper-based devices offer natural advantages, such as screening and separation due to their film-like porous structure, and capillary force for fluid control without external forces.Based on the flow dimensions of the sample in space and the paper base construction, paperbased POCT can be categorized as 1D, 2D and 3D.

| 1D paper-based devices
The structure and function of 1D paper-based devices are presented in a single dimension.These devices allow the sample to react directly on the paper and generate signals in situ.For example, a dot blot POCT. 29Detection systems typically employ optical, electrochemical or electronic techniques to convert the biomolecular process into a readable signal output for rapid detection and diagnosis (Figure 1A).These are the simplest and most basic POC devices. 30

| 2D paper-based devices
The 2D paper-based devices allow lateral or vertical fluid flow, generating a detection signal parallel to this plane.2D paper-based technology offers more possibilities to analyze complex samples due to its larger surface area, higher spatial resolution and ability to integrate multifunctional platforms more easily.There are two main types of 2D paper-based POC assays -lateral flow assays (LFA) 31 and vertical flow assays (VFA). 32he LFA, the most classic and widely used 2D paperbased POC assay, consists of five main components: a supporting base plate at the bottom, followed by the sample pad, the binding pad, the chromatography membrane and the absorbent pad, in that order.The sample pad rapidly absorbs the fluid to be tested, which then flows laterally to the binding pad through capillary tubes; if the fluid to be tested contains the target compound, it will form a complex with the markers of the biologically active material present on the binding pad.This complex then moves along the dialysis membrane to infiltrate the 'test line' and 'control line', which are in turn fixed to the membrane.Chromophores capable of binding to the target complexes are evenly distributed on these lines to allow easy visual interpretation of the detection results (Figure 1B). 33The VFA is based on the stacking configuration and reaction principle of traditional LFA elements.The diffusion flow of the sample from bottom to top takes place stepwise through the individual membranes, allowing the target to react with the membrane alternately (Figure 1C). 34,35

| 3D paper-based devices
The μPAD utilizes the folding, bending and twisting properties of the paper substrate to transform from 2D to 3D, for example, the 3D μPAD. 36This 3D structure facilitates the creation of microfluidic channel interconnections between the different layers of the paper substrate.The introduction of a third dimension (3D) into the structure of paper-based devices can increase the effective sensing area, resulting in increased sensitivity   Abbreviations: AuNP, Au nanoparticle; EVs, extracellular vesicles; GO, graphene oxide; LFA, lateral flow assay; LOD, limit of detection; SiO2@Au, Au wrapped in SiO and the ability to detect multiple targets, leading to more valuable POC detection (Figure 1D). 37,38 3 |TWO-DIMENSIONAL FNAS

INTEGRATED INTO PAPER-BASED POC ASSAYS
The two-dimensional structure of FNAs refers to nucleic acid molecules that are fiber-like and depend on their base sequences to perform their functions, such as linear DNA, hairpin DNA and peptide nucleic acid (PNA).Distinctive screening techniques have shown that dsDNA can selectively capture non-nucleic acid targets such as doxorubicin. 39,40Hairpin DNA refers to an ssDNA molecule that forms a hairpin-like configuration by folding into a stem and a loop.2][43] PNA is a labmade version of DNA linked by peptide bonds.Its backbone structure falls between the peptide bonds of polypeptides and the phosphodiester bonds of nucleic acids, which increases PNA's structural stability and prevents it from being degraded by nucleases and proteases. 44,45 3.1 | 1D paper-based POC assays Linear DNAs are widely applied between different devices (Figure 2).However, due to their simple functionality, they present a challenge when used independently in POC assays.Molecular docking and other simulation techniques have led to the explicit design of linear DNA probes to function as stand-alone recognition elements. 46sing molecular docking, Hassani et al. discovered the stable binding pattern of the cancer drug doxorubicin between the adenine/guanine base pairs of doublestranded DNA (Figure 3A). 47Furthermore, Pomili et al. used this recognition model to achieve a limit of detection (LOD) of doxorubicin as low as 1 ng by using a dsDNA designed to recognize and capture doxorubicin in 1D paper-based assays. 48

| 2D paper-based POC assays
The 2D paper-based devices show better detection performance in terms of sensitivity, specificity and time to analysis of samples.However, the detection of RNA viruses requires additional reverse transcription, which can cause cross-reactivity and result in non-specific reactions.
Because of its versatility and ability to specifically recognize complementary strands, ssDNA has been used to perform multiplexed assays.Yu et al. developed an LFA device to detect SARS-CoV-2 in as fast as 2 min at 30°C, with an LOD of 10 gene copies per test.The device employs an ssDNA probe to replace the capture antibodies of conventional paper-based devices.This device facilitates the simultaneous detection of the RdRp, ORF3a and N genes of the SARS-CoV-2 virus, which are amplified by RT-PCR (Figure 3B). 49Nevertheless, the steps involved in nucleic acid amplification require more sophisticated instrumentation and longer testing times, which limit the implementation of POCT.
To perform multiple simultaneous tests, Phillips et al. developed a technique called the internal splint-pairing expression-cassette translation reaction (INSPECTR).This method can accurately quantify nucleic acids at room temperature without nucleic acid amplification.This technique uses the target specificity of DNA probes to create a cell-free expression system for reporter proteins.The splintR DNA ligase and DNA polymerase allow flexible design of the reporter protein.Multiplexed assays of five respiratory virus RNA targets were successfully achieved simultaneously on LFA (Figure 3C). 50However, the incompatibility of the individual biochemical reactions prevents the assay step from being shortened.On the other hand, reducing the duration of individual reactions reduces the sensitivity of the assay.FNAs can improve sensitivity by building on the classic sandwich method used in immunology.][53][54] Wang et al. developed a commercial diagnostic kit for SARS-CoV-2 infection that was approved by the National Institute of Medicinal Products.The device was able to distinguish SARS-CoV-2 from other infectious diseases using a special and long liner DNA.The long linear DNA probe in this strategy binds to the target to form a S9.6 antibody-probe complex-FNP-labelled S9.6 antibody sandwich structure with high affinity to three S9.6 antibody-specific binding sites.The LOD is 500 copies/mL and is unaffected by clinical sample components. 55ncorporation of specific amplification methods is necessary to further increase the sensitivity of POCT.The process of using T7 nucleic acid exonuclease to apply PNA-assisted target amplicon recognition to recombinase polymerase amplification (RPA) kits and commercial LFA papers is referred to as RPA-TeaPNA-LFA. 56The LOD for SARS-CoV-2 RNA using RPA-TeaPNA-LFA is 6.7 copies/ μL.Targeting two genes (orf2ab and N gene) resulted in 100% specificity and 71.4% sensitivity, respectively.Furthermore, Kim et al. combined hairpin DNA and linear DNA, which are responsible for recognizing and capturing nucleic acids, and were able to form a DNA barcode visible to the naked eye on an LFA device, which led to the successful detection of miR-92a and miR-141 overexpression in exosomes and offered a new method for adjuvant diagnosis of colorectal cancer. 57Similarly, Lamprou et al. detected MiR-21 and miR-let-7a in urine to diagnose bladder and prostate cancer. 58CR was used as an efficient signal amplification strategy for the sensitive detection of various analytes.Specific fragments of Salmonella 16S rRNA were successfully detected using HCR in LFA with a LOD of 1.76 pM.This LOD is approximately two times lower than that without HCR.The LOD for Salmonella was 3 � 10 3 cfu/ mL. 59In further studies, Peng et al. combined HCR with multisite nucleic acid exonuclease III (Exo-III).After integrating the LFA fluorescence signals, the LOD for the 16S rRNA fragment increased to tens of fM, while the limit for three types of bacteria, including Salmonella, increased to tens of cfu/mL (Figure 3D). 60The successful performance of HCR is highly dependent on the precise design of the hairpin DNA sequences.The presence of Reproduced with permission. 37Copyright 2021, Elsevier.complex secondary structures within the target DNA can hinder hybridization, resulting in failure of the HCR.Saisuk et al. addressed this problem by designing two helper oligonucleotides.These oligonucleotides linearized the folding structure of the target using a strandshifting mechanism, allowing the HCR hairpin DNA to access the target site with less difficulty. 61e CHA amplified the detection signals by triggering the next round of the reaction by displacing the target nucleic acid strand; this mechanism is similar to that of a catalyst. 62Detection of hepatitis C virus was achieved using a combination of CHA and LFA with LOD of 1 fM.This method also demonstrated high specificity for the detection of various mismatches. 63Moreover, Mao et al.

F I G U R E 3 Two-dimensional FNAs in paper-based POC assays. (A)
The molecular docking study revealed an intercalation reaction between ds-DNA and doxorubicin.Using dsDNA probe recognize and capture doxorubicin.Reproduced with permissions. 47,48Copyright 2021, MDPI and Copyright 2022, MDPI.(B) Using ssDNA probe for the detection of multiple SARS-CoV-2 genes by LFA.Reproduced with permission. 49Copyright 2020, American Chemical Society.(C) Multiplexed detection of synthetic RNA targets representing five respiratory pathogens using linear DNA probes by INSPECTR.Reproduced with permission. 50Copyright 2023, Springer Nature.(D) HCR combined with Exo-III detects three bacteria and their 16S rRNA fragments by LFA.Reproduced with permission. 60Copyright 2023, American Chemical Society.(E) HCR-based 3D folding card device to detect SARS-CoV-2.Reproduced with permission. 65Copyright 2023, American Chemical Society.(F) Detection of known mutations in SARS-CoV-2 variants using a specially designed TEprobe on a 3D origami device.Reproduced with permission. 66Copyright 2022, Springer Nature.
successfully performed an ultrasensitive detection of nonsmall cell lung cancer-associated miRNAs in urine using hairpin DNA-modified gold nanocages by coupling CHA technology with surface-enhanced Raman spectroscopy (SERS) based on LFA.They reached LODs as low as 3.31 pM for miR-21 and 2.18 pM for miR-196a-5p.This offers a novel possibility for early cancer screening. 64

| 3D paper-based POC assays
The 3D paper-based devices, designed in different dimensions to control reaction times and steps, are closer to a complete paper-based laboratory.Schulte et al. designed a paper folding device based on HCR.This device is similar to opening and closing a book.The left side of the device contains a three-channel membrane and a coreabsorbent pad, while the right side has three conjugate pads and a sample pad.After sample addition, a threechannel disposable device automatically transports the sample in three steps to the test area.The antibody currently in use, which targets the nuclear capsid protein of SARS-CoV-2, achieved an LOD of 2 copies/μL (Figure 3E). 65n enzymatic colorimetric assay called MARVE (for multiplexed, nucleic acid-amplification-free, singlenucleotide-resolved viral evolution) has been developed by Zhang et al. based on a toehold exchange DNA probe (TEprobe).Its main advantage is its ability to detect SARS-CoV-2 while distinguishing specific mutations.Each test has a minimal cost of approximately $0.3 and can be read on-site within 30 min 66 The TEprobe is an integral part of the MARVE and precisely controls the thermodynamic energy of the reaction caused by viral RNA-induced strand displacement in the sample.This control leads to the identification of mutations present in SARS-CoV-2 variants (α, β, γ and δ).The RNA of the mutant virus efficiently triggers the chain-replacement reaction, resulting in the release of Ag(I) ions from the TEprobe, thereby controlling the cleavage of urea, the amount of ammonium ions produced and the pH.Incorporation into folded paper strips allows visual identification of known mutations in the SARS-CoV-2 variant and measurement of pH changes using colorimetric readings on a smartphone (Figure 3F).

| THREE-DIMENSIONAL FNAS INTEGRATED INTO PAPER-BASED POC ASSAYS
Three-dimensional FNAs refer to the active structural domains or conformations of nucleic acid molecules that are essential to their performance, such as aptamers, 67 DNAzymes, 68 G-quadruplexes 69,70 and triplex DNA. 71hey are widely used as recognition and amplification elements for the detection of various proteins, 72,73 nucleic acids, [74][75][76] metal ions [77][78][79] and small molecules. 75nown to scientists as 'antibodies', aptamers are small sequences of oligonucleotides that have been screened in vitro. 80The ability of aptamers to bind tightly to nonnucleic acid ligands, such as proteins and metabolites, and to discriminate between structurally similar substances, further broadens the range of targets that can be detected using functional nucleic acids. 81DNAzymes, also known as deoxyribonucleases, are a synthetic class of DNA oligonucleotides. 68A typical DNAzyme consists of three primary elements in its secondary structure: a cleavage site, a catalytic core and substrate-binding arms. 82These components work with metal ions to cleave RNA that binds to their arms.Compared with enzymes composed mainly of proteins or RNA, DNAzymes are smaller, more chemically stable and can be conveniently and automatically synthesized.G-quadruplexes are tandem repeats of guanine (G)-rich DNA or RNA.They adopt a multiplanar conformation due to Hoogsteen hydrogen bonding. 83G4/ hemin is an exemplary model for catalyzing redox reactions as an artificial enzyme or catalyst. 84Triplex DNA is formed by Hoogsteen interactions between homopurine and homopyrimidine from double-stranded DNA and a single-stranded oligonucleotide. 85Triplex DNA can act as a recognition element and structural switch, facilitating signal transduction.

| 1D paper-based POC assays
The 1D paper-based device is the simplest and most basic type of POC assay available.Yu et al. developed a 1D paper-based ultrasensitive photoelectrochemical (PEC) POC assay using antibodies and tin-doped tin oxide nanotubes exposed to visible light.The platform detects alpha-fetoprotein (AFP), a key diagnostic biomarker for liver cancer, particularly hepatocellular carcinoma.Sndoped SnO 2-x with high photoconversion efficiency was synthesized and co-deposited with AuNPs on the surface of cellulose fiber networks.In addition, the antibody was attached to composite Sn-doped SnO 2-x nanomaterials with good photoconversion efficiency.The electrodes modified with Sn-doped SnO 2-x can create a sensitive steric effect when the target is recognized and bound, resulting in a detection signal based on the reduced photocurrent, allowing sensitive electrochemical detection of AFP as low as 3.84 pg/mL. 86However, antibodies, as biologically active macromolecules, suffer from stability issues, high preparation and production costs and limited specific molecule detection when used in POC assays.
Aptamers can potentially replace antibodies.Azuaje-Hualde et al. developed a 1D dot blot paper-based POC assay for the rapid and sensitive detection of DNA using a hemin-assisted DNAzyme and ABTS redox chromogenic system. 87The hemin-assisted DNAzyme binds specifically to the nucleic acid target with its two arms.This binding then activates its peroxidase-like activity, which catalysis the redox chromogenic system of H 2 O 2 and ABTS (for 2,2diazo-di (3-ethylbenzothiazole-6-sulfonic acid) diamine salt), resulting in a color change from colorless to green or blue.The detection platform achieved visual detection of the Y amelogenin gene fragment within 5 min with the naked eye at a sensitivity of 143 ng.Additionally, quantitative detection was achieved using image analysis with a sensitivity of 45.7 ng (Figure 4A).Although inexpensive and easy to design and operate, 1D paper bases have limited design space, which severely limits their detection performance.

| 2D paper-based POC assays
In traditional antibody-based LFA, there are two main formats: Sandwich and Competitive. 88,89Encouraged by the potential of aptamers to replace antibodies in POC assay design, a competitive paper-based POC platform for the rapid detection of serum CA125 (for cancer antigen 125) was developed by Tripathi et al. by integrating aptamer-gold nanozyme into LFA. 90Serum CA125 can aid in the diagnosis of ovarian cancer.Competitive aptamer binding between CA125 conjugated to gold nanozyme and serum CA125 in the detection zone.The captured gold nanozyme catalyzed the oxidative color development of the DAB/H 2 O 2 substrate.There was a negative correlation between the color intensity and the concentration of CA125 (Figure 4B).The assay is highly specific with an LOD of 3.71 U/mL and a detection time of 20 min.Additionally, the manufacturing cost per strip is less than $1.Although the design is effective in detection, there are bound to be challenges with reagent storage, and the competitive analytical approach may hinder the detection of trace targets.
To address these issues, Climent et al. developed a rapid electrochemiluminescence (ECL) paper-based POC platform for the detection of penicillin in milk using aptamer gated indication (gAID) coupled to glass fiber paper coated with mesoporous silica material. 91In this strategy, the aptamer binds covalently to the mesoporous silica scaffold and further achieves pore closure through non-covalent electrostatic interactions with its surface amino groups.Penicillin induces the aptamer to form a specific motif, which in turn leads to the opening of a pore.The subsequent result of the pore opening is the release of the ECL reporter protein Ru(bpy)  92 The complexity of the electrochemical equipment and the need for pretreatment to remove the effects of interfering matrices on the platform are significant limitations for its POC applications.To avoid this issue, Ulloa-Gomez et al. employed the specific aggregation of aptamer-functionalized polystyrene (PS)-AgNPs and PS-AuNPs particles for two types of detection of trace mercury (Hg 2þ ): colorimetric and electrochemical. 93Lin et al. used aptamers in a dualchannel design to capture fluorescence images and analyses the corresponding RGB (for red, green and blue) values using a smartphone, allowing simultaneous detection of two targets. 94he expanding range of aptamers has led to the development of strategies for the detection of a wider range of targets.The aptamer Ery_06 was screened for erythromycin using the systematic evolution of ligands by exponential enrichment (SELEX) system and combined with recombinase polymerase amplification (RPA) and LFA by Du et al.This platform can detect erythromycin at a concentration of 3 pM in 15 min 95 Subsequently, Yang et al. reported an aptamer with high affinity for the SARS-CoV-2 Omicron variant RBD called SCORe. 96Chen et al. reported an aptamer that can distinguish between Omicron and Delta variants, and they also applied it to LFA. 97 98 Vibrio parahaemolyticus 99 and Ochratoxin A, 100 respectively, using specific aptamers for recognition.
In response to the need to detect multiple targets in complex samples, strategies have been developed based on multi-channel paper-based devices and intelligent signal read-out devices.Jin et al. developed a portable device that uses upconverted fluorescence signals and circular paper discs of aptamers for multiplexed monitoring of water quality. 24Multiple NaYFig 4 :Yb, Er conjugated aptamers and probes were attached in different orientations to a circular paper disc.Samples were added to the disc center, where a specific aptamer captured them, resulting in fluorescent signals.These signals were transmitted through a compact smartphone-based reader that allowed the simultaneous detection of six contaminants in water.The LODs of the device were 115 cfu/mL for Salmonella, 3 ng/mL for Ochratoxin A and Microcystin-LR, 20 nM for Hg 2þ , and 4 nM for Pb 2þ (Figure 4C).Moreover, Tong et al. developed a three-channel mCD-μPAD aptasensor and portable detection device in collaboration with a smartphone.The aptasensor featured covalently modified aptamer fluorescent carbon dots integrated on the surface of MoS 2 nanosheets and was constructed on the 2D sectoral plane.The smart sensor can detect sulfamethazine (SMZ), oxytetracycline (OTC) and chloramphenicol (CAP) with sensitivities of 0.47, 0.48 and 0.34 ng/mL, respectively, in just 15 min (Figure 4D). 101hen further combined with a non-enzymatic cascade amplification reaction, the sensitivity of the paper-based aptamer devices is also enhanced.Chen et al. developed a visually intelligent detection technique using OR and logic gates to identify two types of polychlorinated biphenyls (PCBs) (PCB77 and PCB72).This was achieved by integrating aptamers and Zn 2þ -assisted DNAzymes via toehold-mediated DNA strand displacement. 102In this strategy, the PCB binds specifically to the aptamer and the DNAzyme sequence (DNA1), which is blocked by DNA2.Subsequently, DNA2 dissociates from the complex and DNA1 forms the catalytically active conformation of the DNAzyme assisted by Zn 2þ .This conformation then recognizes and binds to the target analyte, thereby initiating toehold-mediated DNA strand displacement and signal amplification.The Au-DNA3 binds sequentially with the generated hairpin DNA (HP) in the conjugate pad of the LFA and the streptavidin (SA)-biotin-DNA4 in the detection region.This results in an enhanced visual signal for sensitive detection of PCB77.Furthermore, the design of DNA1-DNA2 sequences facilitates the realization of OR and logic gates for the smart detection strategy of PCB77 and PCB72 (Figure 4E).
DNAzyme forms a unique catalytic core that precisely recognises and binds to specific substrate molecules, exerting catalytic activity and signal amplification.Wang et al. exploited the co-activation of DNAzyme by Cu 2þ to trigger the specific hairpin DNA cleavage.This enabled the visual detection of Cu 2þ in water at concentrations as low as 2 ng/mL. 103The editable potential of DNAzyme has led to the development of novel and more effective sensing methods.X10-23 DNAzyme was further modified by Yang et al.They split the catalytic core and extended both arms out of the split, allowing simultaneous target recognition and binding, and substrate binding and shearing.The catalytic performance of X10-23 DNAzyme was further enhanced. 104ew DNAzymes are constantly being identified and confirmed, in addition to the use of existing ones.Ali et al. used a DNA library (DL) to bind approximately 10 14 unique sequences to a fluorescent substrate (FS), and after nine rounds of forward selection and a counter-selection every other round, they discovered the RFD-SA6T1 DNAzyme through an in vitro screening process.This DNAzyme can specifically detect Staphylococcus aureus (Sau) and can be coupled to LFA to detect concentrations as low as 10 6 copies/mL of Sau in diluted sputum in 30 min. 105he G-quadruplex is also considered to be a unique type of DNAzyme.The hemin/G-quadruplex has been applied to μPADs by Zhang et al. to create a timing readout paper device for POC quantitative bioassays of serum K þ . 106The combination of K þ and hemin assists in the formation of motifs from G-quadruplex (G4) sequences, activates the peroxidase-like activity of the Gquadruplex and further catalysis the reaction of colorless TMB and H 2 O 2 to produce the blue signal of poly-TMB.The poly-TMB can change the wetting properties of the b-zone from hydrophilic to hydrophobic, resulting in an increase in the time required for the red ink solution to penetrate the paper.Measurement of K þ can be performed by timing the readout with an LOD of 0.49 μM.Later, Wang et al. exploited the ion-assisted structural switching of G4 to develop a LFA for the rapid detection of heavy metal ions, specifically Pb 2þ .This bioassay enabled the detection of Pb 2þ down to 25 nM in just 15 min. 107tilizing the already commercialized POCT platforms, Zhong et al. employed split G-quadruplex/hemin linked to terminal deoxynucleotidyl transferase (TdT) extension, integrated into a pregnancy test strip platform, to identify potential contaminants (namely 1 nM heavy metal ion Hg 2þ , 0.1 nM xenoestrogen bisphenol A, and 0.05 nM antibiotic penicillin) in milk. 108By replacing specific aptamers, the platform can detect other universal targets within the aptamer limit.Nonetheless, the detection system is more complex and faces economic challenges and inconvenient reagent storage.Li et al. addressed these issues by implementing an amplification approach based on cascade nucleic acid amplification technology and a signal transduction strategy utilizing Gquadruplex/hemin. 109aper-based POC assays have also employed triplex DNA and Y-shaped DNA.Huang et al. utilized target-induced molecular switches on triplex DNAfunctionalized carbon nanotubes (CNTs) incorporated into a lateral flow nucleic acid bioassay that can visually detect both nucleic acids and proteins simultaneously.The method can detect at least 25 pM target DNA and 0.25 nM thrombin within 20 min. 110

| 3D paper-based POC assays
Electrochemistry is the most commonly used method in paper-based 3D devices.Li et al. introduced a novel approach in μPAD for dual-signal detection of H 2 O 2 based on a novel dual photoelectrochemical/colorimetric cell analysis.The hydroxyl radical (•OH) can cleave DNA in situ.The optical signals from the quantum dots are combined with the visual signals obtained from color conversion of the •OH and TMB.The 3D paper-based folding device integrates the method and enables sensitive and real-time visual detection of H 2 O 2 released from MCF-7 cells. 112However, this platform is limited to the detection of H 2 O 2 , making it difficult to achieve universal detection of other targets.
To address this issue, Wang et al. developed a paperbased aptasensor.They utilized an anti-EGFR (for epidermal growth factor receptor) aptamer to bind to the surface of a graphene-modified electrode to detect EGFR in a small sample volume without any labeling.In this approach, a 3D origami paper functions as a valve between sample introduction and detection, reducing the sample volume.Amino-functionalized graphene oxide (NH 2 -GO)/thionine (THI)/gold particle (AuNP) nanocomposites were used to modify the working electrode, generating electrochemical signals and enabling the anchoring of the aptamer to its surface.The aptamer specifically identifies and captures EGFR, leading to changes in the electrochemical signals.The aptasensor can detect EGFR within 60 min with a sensitivity of 5 pg/mL. 113ubsequently, Jiang et al. used a comparable 3D origami electrode and reported that an aptamer-modified black phosphorus nanosheets (BPNSs) sensing probe electrodeposited on a microfluidic origami paper-based electrode surface could detect the peanut allergen Ara h1, with a detection time of 20 min and a LOD of 21.6 ng/mL (Figure 4F). 114

FNAS INTEGRATED INTO PAPER-BASED POC ASSAYS
The conjugation of different FNAs, including linear DNA, aptamers, triplexes and DNAzymes, with different nanomaterials, such as metal nanomaterials, carbon nanotubes and bio-nanomaterials, results in the generation of composite nanomaterials, such as aptamer-AuNP, 115 linear DNA-carbon nanotubes 116 and hairpin DNA functionalized Fe 3 O 4 magnetic nanoparticles. 117These composite nanomaterials exhibit unique or enhanced functional properties due to the synergistic activity of the two components.Previous research has focused on the development of active structures that can dynamically adapt for applications such as biosensing, drug delivery, molecular logic, molecular electronics and other areas. 118,119

| 1D paper-based POC assays
The 1D paper-based devices are the most basic and straightforward.However, their simple construction results in a smaller surface area, which significantly hinders the development of POC assays based on 1D paperbased devices.To date, no studies have integrated threedimensional FNA nanomaterials with complex and robust designs into 1D paper-based devices.

| 2D paper-based POC assays
The hybridization reaction using the DNA1-target-DNA2 'sandwich' technique has a wide range of applications in LFA.An LFA for visual detection of DNA single base mismatches with high specificity was reported by He et al.The strategy is based on sulfhydryl-modified hairpin oligonucleotides (HOs) that can bind to dual-target DNA via their sulfhydryl groups on the surface of gold nanoparticles (Au-NPs).Upon specific binding to the target, the HOs unfold and expose stem fragments.The exposed stem loops hybridize with a DNA probe immobilized in the detection zone of the test strip, resulting in the formation of a red band on the test line via the deposition of gold nanoparticles. 120n this basis, Qiu et al. developed a multi-walled carbon nanotube (MWCNT)-based lateral flow bioassay for the sensitive and rapid detection of DNA sequences using a "sandwich" DNA hybridization reaction between a nucleic acid target covalently bound to a DNA probe in a carbon nanotube (CNT) and a DNA probe immobilized in the detection zone of LFA.The assay uses a "sandwich" DNA hybridization reaction between a nucleic acid target covalently bound to a CNT and a DNA probe immobilized in the detection zone of LFA, which produces a black band on the test line due to carbon nanotube deposition (Figure 5A). 121Yao et al. then employed this method to detect Hg 2þ in water. 122n addition, Zhang et al. developed a low-cost and simple fluorescent POC assay based on Cy5-labelled functional ssDNA conjugated to a graphene oxide surface (ssDNA-GO) for the detection of the heavy metals Hg 2þ , Ag þ and residues of aminoglycoside antibiotics in food in a multiplexed assay, with LODs in 10 min of 121 nM, 47 and 153 nM, respectively (Figure 5B). 123Xu et al. employed a PtAu@CNTs nanocomposite catalytic chromogenic signal amplification strategy based on the "sandwich" DNA hybridization reaction to develop an LFA capable of detecting nucleic acids with an ultra-low detection limit of 0.43 pM. 124Fu et al. further developed a SERS nucleic acid assay using a similar "sandwich" DNA hybridisation reaction based on AuNP, lowering the LOD to 0.24 pg/mL. 125  Reproduced with permission. 130Copyright 2021, Springer Nature.(F) A CASLFA platform based on a novel bivalent aptamer and CRISPR/ Cas signal transduction for small molecule detection.Reproduced with permission. 135Copyright 2022, Elsevier.(G) A 3D lab-on-chip device based on MOF-ssDNA and CHA for Zn 2þ ultra-sensitive detection.Reproduced with permission. 139Copyright 2019, American Chemical Society.
To further enhance the detection sensitivity for trace targets, Gao et al. developed a three-dimensional DNA-AuNPs network amplification technique and introduced it into LFA to construct an ultra-sensitive POC assay for nucleic acid detection.This method employs ssDNA modified with sulfhydryl and biotin at the other end.At one end, the ssDNA is immobilized on surface of AuNPs using sulfhydryl, while at the other end it binds to streptavidin-coated gold nanoparticles (Au-NPs) via biotin.Biotin binding results in a large number of gold nanoparticle (AuNP) aggregates, which are then anchored to the test line by a sandwich strategy for LFA.The aggregates are attached to the test line in LFA as an ultra-sensitive method for the detection of nucleic acids with a LOD of 0.01 pM (Figure 5D). 127nspired by the three-dimensional DNA-AuNPs network amplification technique, Velu et al. have developed interlocking inversion assembly-based nanoaptasensors (LIANAs) for ochratoxin A detection using aptamers. 128Moreover, Tian et al. combined a DNA-AuNPs network three-dimensional structure amplification technique with a terminal deoxynucleotidyl transferase sandwich assay to identify the antimicrobial drug enrofloxacin (ENR).The platform is capable of detecting ENR at an LOD of 0.1 μg/L in 5 min, preventing the antibiotic abuse. 129The paper-based POC platform demonstrated improved sensitivity by combining HCR, entropy-driven strand replacement, and other enzymefree thermostatic amplification techniques.Li et al. reported a portable and highly sensitive platform for the detection of extracellular vesicles (EVs) carrying glypican-1 (GPC1) mRNA, which is associated with early pancreatic cancer.The platform used a novel two-step amplification method known as Catalytic Hairpin Assembly and Gold-Enhanced strips (CHAGE).The CHAGE assay has a LOD for mRNA of 100 fM (Figure 5E). 130mplementation of an electrochemical detection platform enhances the precision detection capabilities of paper-based POC assays.A paper-based bipolar electrode electrochemiluminescence platform for the detection of multiple miRNAs was developed by Wang et al.The platform is based on a three-dimensional DNA nanomachine model and a strategy of cycling the target miRNA, resulting in double amplification of electrochemiluminescence signals.The platform is capable of simultaneously detecting miRNA-155 and miRNA-126 with LODs of 5.7 fM and 4.2 fM, respectively. 117etection of microbial nucleic acids alone can lead to false positives, so there is a need to detect non-nucleic acid biomolecules, including proteins.Aptamers demonstrate excellent recognition in the detection of non-nucleic acids.To this end, Kim et al. identified two aptamers, J3APT and JH4APT, which selectively target different sites on H5N2 avian influenza virus particles.They then applied an AuNP-based sandwich signaling technique to LFA to accurately detect whole avian influenza H5N2 virus particles.The LOD was 6 � 10 5 /mL. 131Moreover, Somvanshi et al. screened aptamers for E. coli O157:H7 and Salmonella typhimurium, respectively, which were then immobilized on the surface of gold nanoparticles.They were applied to a multi-channel sensing system using microfluidic paper.The platform demonstrated high sensitivity, detecting E. coli O157:H7 and Salmonella typhimurium at 10 3 cfu/mL and 10 2 cfu/mL, respectively. 132Inspired by phage-host bacterial interactions, Zhang et al. created a recombinant protein called STFP, which consists of a SUMO tag and a phage tail fiber protein.The protein binds specifically to the AuCoNPs-aptamer and induces a "sandwich" conformation, resulting in signaling.As a result, a rapid and sensitive LFA was developed that is capable of detecting pathogenic bacteria at the sensitivity of 48 cfu/mL. 133he target induces the aptamer to form the correct structure, resulting in the separation of the aptamer bound to the AuNPs from its surface.Therefore, Alnajrani performed a "TURN OFF" assay for dabigatran etexilate in blood with LFA. 134However, the sensitivity is unsatisfactory, mainly due to the "TURN OFF" detection strategy and the lack of powerful amplification.To address this issue, Li et al. used a novel bivalent aptamer as the recognition element and combined it with a CRISPR/Cas signaling and amplification system on LFA.As a result, a CRISPR/Cas12a-mediated aptamer lateral flow assay (CASLFA) platform was developed that can visually indicate the presence of ATP with a "TURN ON" signal in just 26 min.The platform achieved an excellent LOD of 0.85 μM (Figure 5F). 135ioassay development is not only limited to sensing strategies but also includes the exploration of smart nanomaterials and equipping POC assays with smart response characteristics.Cherkasov et al. developed an ultrasensitive smart nanoparticle beacon with switchable affinity for biomedical targets based on a smart nanoagent combining gold nanoparticles with a low-energy polymer structure.The on/off switch of this nanoparticle beacon can be coupled to LFA, enabling ultrasensitive detection of DNA down to 30 fM in 15 min (Figure 5C). 136ligonucleotides containing artificial nucleobases and pseudo-complementary sequences reduce duplex formation in pseudo-complementary pairs while maintaining duplex formation in target (complementary) oligomers.This mechanism allows oligonucleotides to penetrate double-stranded DNA.López-Tena et al. integrated mixed sequence invasion and RT-RPA (for reverse transcriptionenzymatic recombinase amplification) response to pseudo-complementary G:C base pairs applied to LFA. 137 GAO ET AL.

| 3D paper-based POC assays
The 3D paper-based devices can incorporate additional amplifying elements such as nanoparticles or fluorescent dyes to enhance signal amplification.Li et al. fabricated 3D hollow-channel origami electrodes and developed a lab-on-chip device to detect Zn 2þ with high sensitivity.The device is based on three-dimensional composite nanomaterials consisting of porous metal-organic frame-(MOFs) and oligonucleotide chains attached to their surfaces. 138The device employs a three-step amplification strategy to enable the sensitive detection of Zn 2þ ions.In step I, 3D hollow-channel folded paper electrodes with oxidized graphene are deposited to enhance the adhesion of the (Fe-P)n-MOF and improve the conductivity.In step II, rolling circle amplification (RCA) is used to efficiently amplify the G-quadruplex, while in step III, MOF is used to enhance the catalysis of hemin/ G-quadruplex on H 2 O 2 .The detection of Zn 2þ is achieved using capture probes to ensure specificity.On this basis, Sun et al. reported an ultrasensitive detection platform for glycoproteins based on 3D origami electrodes and three-dimensional nucleic acid nanocomposites (Figure 5G). 139

| CONCLUSIONS AND PERSPECTIVES
Paper-based assays are economical and simple platforms.Functional nucleic acids selectively recognize and bind to target molecules, amplifying and transducing the signal.Thus, they can be utilized in monitoring various analytical targets, including biomolecules, environmental pollutants, and disease markers.In this review, we provide a comprehensive overview of FNAs and paper-based devices.Subsequently, the representative applications of various FNAs are discussed in paper-based devices from the perspective of the three spatial dimensions.
Improving detection sensitivity, enabling multi-target detection, monitoring changes in the target molecule concentration in real time, visualizing output results and simplifying operation are key issues in POC assay development.The properties of biomolecular recognition and signal transduction have facilitated bioassay development, driven technological innovation and expanded the applications of paper-based devices.However, several pressing issues need to be considered before the use of bioassays can be implemented in real-world scenarios.Firstly, current paper-based POC assays lack sufficient sensitivity, severely limiting their ability to detect trace levels.Secondly, the detection of multiple target combinations is challenging due to the limited range of recognizable targets.Finally, current paper-based POC assays are mainly focused on the development of 2D paper-based devices.The simple design and layout of 2D paper-based devices pose challenges for achieving multifunctional integration.The 3D paper-based devices offer the additional space and channels required to accommodate multiple functional elements for the simultaneous detection of multiple target molecules.The 3D paper-based devices offer a larger reaction surface area, faster reaction speed, stronger signal amplification and improved sample handling.Nevertheless, challenges can arise due to preparation complexity, mass transfer limitations, and factors such as reproducibility and stability.Currently, the development of paperbased 3D devices is in its infancy, with significant opportunities for further exploration.The challenges associated with the development of paper-based devices are expected to be overcome by fine-tuning materials, producing more powerful FNAs and advancing interfacial modification technologies for paper-based devices.

T A B L E 1 FNA
Comparison of paper-based FNAs integrated into paper-based POC assays., and Sty as 0.

F I G U R E 1
Schematic diagram showing DNA nanostructurebased paper POCT.Functional nucleic acids (FNAs) used in paper-based devices are mainly 2D, 3D and nanomaterial conjugated FNAs.These FNAs are integrated into 1D, 2D and 3D paper-based devices to develop a diversity of POC assay platforms with high detection performance.

F I G U R E 2
The 1D, 2D and 3D paper-based POCT devices.(A) Scheme of the 1D paper-based dot POCT device.Reproduced with permission. 30Copyright 2015, Elsevier.(B-C) Schematic representation showing the LFA (B) and VFA (C) device.Reproduced with permissions. 33,140Copyright 2021, Wiley-VCH GmbH and Copyright 2019, Elsevier.(D) Schematic illustration of the 3D-μPAD.

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Y-shaped DNA was incorporated into an endonuclease Nt.BbvCI-based assay circuit by Huang et al.The circuit employs a lateral flow paper base with dual test lines to detect microRNAs.The sensitivity is 0.1 pM miR-16 in water. 11110 GAO ET AL.

F I G U R E 5
Nanomaterial-conjugated FNAs integrated into paper-based POC assays.(A) Schematic representation showing a MWCNT-based LFA for the sensitive and rapid detection of DNA.Reproduced with permission. 121Copyright 2015, Elsevier.(B) A simple fluorescent POC device based on ssDNA-GO for multiplexed assays.Reproduced with permission. 123Copyright 2015, Elsevier.(C) Schematic illustration of the LFA that relied on nanoparticle beacon on/off switches bound to linear DNA for ultrasensitive DNA detection.Reproduced with permission. 136Copyright 2020, American Chemical Society.(D) Nucleic acid ultra-sensitive LFA platform integrated DNA-AuNPs network amplification strategy.Reproduced with permission.Copyright 2017, Elsevier.(E) Schematic illustration of the LFA based on CHAGE, which used target-triggered CHA and gold-enhanced technology, to detect mRNA in EVs.
Furthermore, Jin et al., Ying et al. and Liu et al. performed LFA for the detection of tobramycin, 126these basis, Wang et al. designed a SERS-based LFA with two test lines and a control line for the simultaneous detection of two nucleic acid targets.126