Rolling Circle Amplification Reaction Research Articles

Endonuclease Q as a robust enhancer for nucleic acid amplification

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

A photothermal aptasensor based on rolling circle amplification-enriched DNAzyme for portable detection of ochratoxin A in grape juice

Aptasensors for detection of ochratoxin A (OTA) have been extensively studied, but the majority of them require costly and large-scale equipment as signal readers. Herein, a photothermal aptasensor capable of portable detection of OTA through a thermometer was developed on basis of aptamer structural switching and rolling circle amplification (RCA)-enriched DNAzyme. Oligonucleotides and alkaline phosphatase (ALP) modified magnetic beads were prepared. The binding of aptamers to OTA led to the release of ALP labeled complementary DNA. After magnetic separation, ALP catalyzed the padlock dephosphorylation, inhibiting the subsequent RCA reaction. This process converted the OTA concentration into the amount of the photothermal reagent oxTMB produced from the catalytic reaction induced by RCA-enriched DNAzyme. Under the optimal conditions, the detection limit (LOD) of this aptasensor was 2.28 nM in a clean buffer, while the LOD reached 2.43 nM in 2 % grape juice. The good performance of the photothermal aptasensor makes it possible to measure OTA pollution in low resource environments.

A surface-enhanced Raman scattering and colorimetric dual-mode aptasensor for ultrasensitive detection of kanamycin based on DNA hydrogel network fishing the MIL-101@AuNP nanohybrids

It is of utmost importance to develop a simple, sensitive, and dependable technique for detecting kanamycin (Kana). Utilizing MIL-101@AuNP nanohybrids and DNA hydrogel, we devised a dual-mode aptasensor for Kana detection employing surface-enhanced Raman spectroscopy (SERS) and a colorimetric method. The MIL-101@AuNP nanohybrids displayed excellent peroxidase-like and SERS activities. In the existence of Kana, the aptamer specifically bound to Kana, releasing complementary DNA strands (cDNA). Employing free cDNA as a primer, circular DNA templates were generated for the rolling circle amplification (RCA) reaction. DNA hydrogel network formed via the physical entanglement and hybridization of two ultralong single-strand DNA chains from the double RCA-generated, capturing MIL-101@AuNP nanohybrids, thereby enhancing the SERS signal of the DNA hydrogel. Concurrently, the absorbance signal of 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system catalyzed by unfished MIL-101@AuNP was weakened. The dual-mode aptasensor, employing SERS and colorimetric methods, quantitatively detected Kana within concentration ranges of 0.003–300 ng/mL and 0.03–100 ng/mL, respectively, with detection limits (LOD) of 0.001 ng/mL and 0.018 ng/mL, respectively. Importantly, this method was applied to detect Kana-spiked milk samples, yielding satisfactory spiked recoveries. The proposed approach combines the advantages of SERS and colorimetric methods, offering high sensitivity, reliability, and broad applicability, thereby presenting a novel thinking for constructing multimode sensing platforms.

Sensitive detection of exosomes by an electrochemical aptasensor based on double nucleic acid amplifications

Exosomes are extracellular vesicles secreted by mammalian cells and recognized as a powerful non-invasive cancer marker because they carry the cellular components specific to parent cells. The development of a convenient, rapid and sensitive exosome detection method is very important for the early diagnosis of cancer. In this study, an electrochemical biosensor based on two signal amplification methods, primer exchange reaction (PER) and rolling circle amplification (RCA), was developed for the high sensitive detection of exosomes. First, exosomes were specifically captured by CD63 aptamer and DNA strands (T) were released through a chain replacement reaction. Then the T strands initiated the PER reaction, producing a large number of short strands of DNA (P). Afterwards, RCA reaction was carried out on the electrode surface. Finally, a large amount of horseradish peroxidase (HRP) was bound to the electrode surface to catalyze o-phenylenediamine (OPD) oxidation, and the reduction current signal was obtained to realize the quantitative detection of exosomes. Under the optimal experimental conditions, there was a good linear relationship between DPV signal and the logarithm of exosome concentration in the range of 88 ∼ 8.8 × 106 particle/μL with a low limit of detection of 22 particle/μL. This strategy could realize sensitive detection of exosomes and provide a new strategy for the detection of other tumor markers.

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Label/immobilization-free Cas12a-based electrochemiluminescence biosensor for sensitive DNA detection

Electrochemiluminescence (ECL) is one of the most sensitive techniques in the field of diagnostics. However, they typically require luminescent labeling and electrode surface biological modification, which is a time-consuming and laborious process involving multiple steps and may also lead to low reaction efficiency. Fabricating label/modification-free biosensors has become one of the most attractive parts for simplifying the ECL assays. In this work, the ECL luminophores carbon dots (CDs) were encapsulated in DNA hydrogel in situ by a simple rolling circle amplification (RCA) reaction. Upon binding of the target DNA, active Cas12a induces a collateral cleavage of the hydrogel's ssDNA backbone, resulting in a programmable degradation of the hydrogel and the release of CDs. By directly measuring the released CDs ECL, a simple and rapid label/modification-free detection of the target HPV-16 was realized. It is noted that this method allowed for 0.63 pM HPV-16 DNA detection without any amplification step, and it could take only ∼60 min for a fast test of a human serum sample. These results showed that our label/modification-free ECL biosensor has great potential for use in simple, rapid, and sensitive point-of-care (POC) detection.

Hyperbranched RCA-mediated construction of a dendritic DNA nanostructure for ratiometric fluorescence biosensing

As a robust isothermal strand amplification technique, rolling circle amplification (RCA) is popularly adopted for constructing various homogeneous fluorescence biosensing strategies. However, the limited functional unit decoration on linear RCA products and the slow homogeneous reaction kinetics usually restrict their performance. Herein, by designing a target biorecognition-triggered hyperbranched RCA reaction to construct an Mg2+-dependent DNAzyme (MNAzyme)-decorated dendritic DNA nanostructure, a novel ratiometric fluorescence biosensing method was proposed. The catalyzed cleavage of MNAzymes toward a short stand labeled with the FAM fluorophore and its quencher realized a “signal-on” fluorescence output. Meanwhile, a “signal-off” fluorescence output of 2-aminopurine (2-AP) labeled at a DNA hairpin was realized through the DNA hybridization with the nanostructure. Besides efficient acceleration of homogeneous reaction kinetics, these fluorescence responses were dramatically amplified by the high decoration of MNAzymes on the dendritic nanostructure and its strong signal suppression toward 2-AP. Together with the exonuclease III-amplified target biorecognition reaction, a very low detection limit of 0.53 fg mL–1 and a six-order magnitude linear range were endowed for kanamycin antibiotic assay. This excellent analytical performance and its high selectivity, and good operability determine promising prospect of the method for practical applications.

Tumor-associated antigen-specific cell imaging based on upconversion luminescence and nucleic acid rolling circle amplification.

Tumor-associated antigen (TAA)-based diagnosis has gained prominence for early tumor screening, treatment monitoring, prognostic assessment, and minimal residual disease detection. However, limitations such as low sensitivity and difficulty in extracting non-specific binding membrane proteins still exist in traditional detection methods. Upconversion luminescence (UCL) exhibits unique physical and chemical properties under wavelength near-infrared light excitation. Rolling circle amplification (RCA) is an efficient DNA amplification technique with amplification factorsas high as 105. Therefore, the above two excellent techniques can be employed for highly accurate imaging analysis of tumor cells. Herein, we developed a novel nanoplatform for TAA-specific cell imaging based on UCL and RCA technology. An aptamer-primer complex selectively binds to Mucin 1 (MUC1), one of TAA on cell surface, to trigger RCA reaction, generating a large number of repetitive sequences. These sequences provide lots of binding sites for complementary signal probes, producing UCL from lanthanide-doped upconversion nanoparticles (UCNPs) after releasing quencher group. The experimental results demonstrate the specific attachment of upconversion nanomaterials to cancer cells which express a high level of MUC1, indicating the potential of UCNPs and RCA in tumor imaging.

An rolling circle amplification-assisted CRISPR/Cas12a-based biosensor for protein detection

Glycated hemoglobin (HbA1c) has been extensively studied as a pivotal marker for monitoring and diagnosing diabetes. In this study, we introduce a highly sensitive and rapid enzyme-catalyzed rolling circle amplification (RCA)-assisted nicked-PAM CRISPR/Cas12a assay (termed “RANPC”). The RANPC system initially recognizes and captures the target using immunomagnetic beads and antibodies, forming a sandwich structure. The antibodies, upon ligating the primers, trigger the RCA reaction, resulting in the generation of single-stranded DNA with numerous repetitive sequences. The product strand of RCA then initiates the trans-cutting activity of nicked-PAM CRISPR/Cas12a, ultimately reporting the target concentration through fluorescent signaling. This study combines dual antibody capture, signal amplification through RCA, and signal output by Cas12a, resulting in high sensitivity, high specificity, and high signal-to-noise ratio. Moreover, the strategic incorporation of an incomplete PAM design in the CRISPR/Cas12a system substantially reduces background interference and enhances assay specificity relative to previous conventional Cas12a detection methods. RANPC possesses advantages such as ultra-sensitivity (with the lowest detection limit up to 0.129 pg/mL and a linear range of 12.8 pg/mL to 1 µg/mL), high specificity, rapid reaction time (approximately 50 min), and straightforward operation. Under optimized experimental conditions, we successfully detected HbA1c in eight real-world blood samples, and the results were in good agreement with the theoretical values. This suggests that this method holds promise as a new approach for the analysis of HbA1c levels in the clinical setting.

Target mediated bioreaction to engineer surface vacancy effect on Bi2O2S nanosheets for photoelectrochemical detection of FEN1

Photoelectrochemistry represents a promising technique for bioanalysis, though its application for the detection of Flap endonuclease 1 (FEN1) has not been tapped. Herein, this work reports the exploration of creating oxygen vacancies (Ov) in situ onto the surface of Bi2O2S nanosheets via the attachment of dopamine (DA), which underlies a new anodic PEC sensing strategy for FEN1 detection in label-free, immobilization-free and high-throughput modes. In connection to the target-mediated rolling circle amplification (RCA) reaction for modulating the release of the DA aptamer to capture DA, the detection system showed good performance toward FEN1 analysis with a linear detection range of 0.001–10 U/mL and a detection limit of 1.4 × 10−4 U/mL (S/N = 3). This work features the bioreaction engineered surface vacancy effect of Bi2O2S nanosheets as a PEC sensing strategy, which allows a simple, easy to perform, sensitive and selective method for the detection of FEN1. This sensing strategy might have wide applications in versatile bioasssays, considering the diversity of a variety of biological reactions may produce the DA aptamer.

Plasmonic Gold Nanostar-Based Probes with Distance-Dependent Plasmon-Enhanced Fluorescence for Ultrasensitive DNA Methyltransferase Assay.

The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO2)-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.

DNA nanoflowers encapsulating horseradish peroxidase as a signal amplification tag for rapid diagnosis in cytology specimens

Rapid analysis of cytology samples obtained by minimally invasive sampling is essential in clinical diagnosis, yet existing diagnostic methods are often cumbersome and time-consuming. To rapidly detect the scanty number of cells in specimens, a multifunctional detection probe based on DNA nanoflower structure was prepared in this study, which possesses both signal output capability of fluorescence and oxidation reaction (colorimetric assay) for ultra-sensitive cancer cell detection. Firstly, DNA nanoflowers encapsulating horseradish peroxidase (HRP-DNA Nanoflowers, HDFs) were prepared by the rolling circle amplification (RCA) reaction and then hybridized with the aptamers labeled with fluorophores to construct the multivalent aptamer HDFs (Multi-Aptamer-HDFs) with fluorescence effect and efficient cancer cell targeting. The Multi-Aptamer-HDFs probe can be used to detect fresh clinical cytology specimens directly for fluorescence observation with sensitivity and specificity of 88.64%, and 84.85%, respectively. In addition, Multi-Aptamer-HDFs has peroxidase activity due to the encapsulation of HRP, which can catalyze the oxidation of substrates such as ABTS and DAB for color development. Therefore, we applied Multi-Aptamer-HDFs to one-step staining of clinical cytology cell blocks with a sensitivity of 88.41% and a specificity of 83.33%, which required fewer steps and reagents with a significantly shorter time required for immunocytochemical staining. The multifunctional probe, Multi-Aptamer-HDFs, prepared in this study is expected to be better applied in the rapid cellular analyses and one-step immunostaining, which is expected to provide a new means for rapid on-site assessment and on-site preliminary diagnosis.

The construction of COFs functionalized CRISPR electrochemical sensor for ultrasensitive detection of bacteria by hyper-branched rolling circle amplification

Rapid, sensitive, and low-cost foodborne pathogens detection is crucial for dealing with foodborne diseases caused by bacteria. In this work, we designed a new CRISPR/Cas9-mediated electrochemical biosensor for ultrasensitive detection of Escherichia coli (E. coli) based on multifunctional covalent organic framework-based nanocomposite (GOx-AuNPs-COF-H2) and hyperbranched rolling circle amplification (HB-RCA) reaction. When specific DNA targets were present in the system, the targets could be recognized and cleaved by CRISPR-Cas9 system, which triggered strand displacement reaction (SDR) and HB-RCA reaction. By introducing GOx-AuNPs-COF-H2 nanocomposite, H2 probe could hybridize with RCA products to bind to the electrode surface. After added glucose solution, GOx could catalyze it in the detection system and thus offered significant current signals for bacteria detection. This approach showed high analytical performance for assay of E. coli with the detection limit (LOD) of 10 CFU/mL. Furthermore, real samples were detected by this approach, demonstrating its promising potential for foodborne pathogens detection in food monitoring and clinical diagnosis.

Highly sensitive detection of Pb2+ in the environment with DNAzyme and rolling circle amplification reaction

Lead (Pb2+) is a toxic heavy metal that can severely pollute the environment and cause harm to public health. Therefore, the prompt and accurate monitoring of lead levels in the environment is vital. In this study, a novel DNAzyme-based cascade signal amplification biosensor that could detect Pb2+ with high sensitivity was designed through the combination of the strand displacement reaction (SDR) and rolling circle amplification (RCA). When Pb2+ is absent, RCA is triggered under the synergistic action of T4 DNA ligase and phi29 DNA polymerase with an artificially fluorophore-labeled S-chains being released to replace the upstream products generated by repeated RCA, thereby restoring the quenched fluorescence and emitting a strong fluorescent signal. After adding Pb2+, 8–17 DNAzyme binds specifically to Pb2+ and catalyzes the cleavage of the rA site on a single-stranded DNA with artificially modified rA site to restrict the RCA. The designed sensor provides a linear detection range for Pb2+ from 25 pM to 1 µM, with a low limit of detection 8.3 pM. Significantly, this sensor still demonstrates satisfactory performance when used for detecting Pb2+ in environment samples (e.g., river water). We consider that our study can provide reference values and ideas for the development of heavy metal ion detection methods.

A cascade signal-amplified fluorescent biosensor combining APE1 enzyme cleavage-assisted target cycling with rolling circle amplification.

A cascade signal-amplified fluorescent biosensor was developed for miRNA-21 detection by combining APE1 enzyme-assisted target recycling and rolling circle amplification strategy. A key feature of this biosensor is its dual-trigger mechanism, utilizing both tumor-endogenous miRNA-21 and the APE1 enzyme in the initial amplification step, followed by a second rolling circle amplification reaction. This dual signal amplification cascade significantly enhanced sensitivity, achieving a detection limit of 3.33 pM. Furthermore, this biosensor exhibited excellent specificity and resistance to interference, allowing it to effectively distinguish and detect the target miRNA-21 in the presence of multiple interfering miRNAs. Moreover, the biosensor maintained its robust detection capabilities in a 10% serum environment, demonstrating its potential for clinical disease diagnosis applications.

Cas-Rainbow: Cas12a-driven single-reaction multiplex detection system

Recently, CRISPR/Cas system has garnered considerable attention owing to its high cleavage efficiency. However, it lacks the ability to differentiate between signal sources. To address this, we have developed a multiplex detection system called Cas-Rainbow. This system combines CRISPR/Cas with rolling circle amplification (RCA) and hybridization chain reaction (HCR) to enable the detection of multiple miRNAs using reporter probes labeled with different fluorescent groups. The RCA product can trigger the formation of HCR and activate the Cas12a/crRNA complex, achieving the simultaneous detection of multiple miRNAs. Detection limits of three miRNAs (miR-155, miR-10b, and miR-21) were 33 fM, 41 fM, and 79 fM, respectively. Cas-Rainbow system could effectively identify these three miRNAs in serum, distinguishing breast cancer patients from healthy donors with accuracy of 100 %. Moreover, this system exhibited exceptional performance in logic gate circuits, showcasing its remarkable versatility and robustness. In summary, our proposed method offers a new approach for multiplex detection based on the CRISPR/Cas system.

Light-Triggered Signal Enhancement Strategy Integrated with a CRISPR/Cas13a-Based Assay for Ultrasensitive and Specific miRNA Detection.

The development of facile, accurate, and affordable assays for microRNAs (miRNAs) in early cancer is greatly desirable but encounters an obstacle due to low cellular abundance in biofuids. In this study, we present a novel approach called a light-triggered exponential amplification strategy coupled with a CRISPR/Cas13a-based diagnostic system (LEXPA-CRISPR), which directly transduces rare miRNA targets into photocontrolled signal enhancement response. This innovative platform leverages trans-cleavage of CRISPR/Cas13a, activated by the miRNA target, to cleave specific RNA fragments within the MB@PC-NAC assembly, thus releasing free PC-single-stranded DNA (PC-ssDNA) that is modified by a photocleavable linker (PC linker). UV irradiation is further employed toward the photoresponsive PC-ssDNA, resulting in instantaneous generation of oligo with a new 5' phosphate group (Pho-ssDNA). The Pho-ssDNA serves as a trigger for rolling circle amplification (RCA) reaction, which generates thousands of long ssDNA repeats of diverse lengths with a strong fluorescence signal. Through optimization, we achieved a detection limit of 1 fM for miR21 without the need for target amplification. Moreover, the programmable versatility of LEXPA-CRISPR is also demonstrated for miR17 determination only with simple modification of CRISPR RNA (crRNA) sequences. This proposed biosensor successfully monitored the levels of miR21 and miR17 in tumor cells, showing a satisfactory consistency with the standard qRT-PCR method. Conclusively, LEXPA-CRISPR represents a promising strategy for ultrasensitive miRNA detection. It combines the advantages of light-triggered signal amplification and robust collateral cleavage activity of Cas13a, making it an attractive tool for practical CRISPR-based diagnostics.

Multivalent DNA Flowers for High-Performance Isolation, Detection, and Release of Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (T-EVs) hold great promise for understanding cancer biology and improving cancer diagnostics and therapeutics. Herein, we developed multivalent DNA flowers (DFs) containing repeated and equidistant EpCAM aptamers for the efficient isolation of T-EVs. The multivalent aptamer chains in DFs had good flexibility to adapt to the surface morphology of T-EVs and achieved multivalent ligand-receptor interactions, thus showing enhanced isolation ability compared to monovalent aptamers. Compared with other materials for isolation of EVs, DFs were generated by rolling circle amplification (RCA) and self-assembled into microspheres in a one-pot reaction, and the recognition molecules (aptamers) were directly replicated and assembled during the RCA reaction instead of chemical modification and immobilization on the surface of solid materials. Moreover, as optically transparent biomaterials, the content of EpCAM+ EVs could be directly reflected via membrane-based hydrophobic assembly of signaling modules in DFs@EpCAM+ EVs complex, and we found that the amount of EpCAM+ EVs showed greater accuracy in cancer diagnosis than total EVs (88.3 vs 69.7%) and was also higher than the clinically commonly used marker carcinoembryonic antigen (CEA) (88.3 vs 76.7%). In addition, T-EVs could be released by lysis of DFs with the nuclease, gently and easily, keeping high intact and activity of EVs for downstream biological function studies. These results demonstrated that DFs are efficient and nondestructive tools for isolation, detection, and release of T-EVs.

Precise Detection of Viral RNA by Programming Multiplex Rolling Circle Amplification and Strand Displacement.

Detection of viral infections (e.g., SARS-CoV-2) with high precision is critical to disease control and treatment. There is an urgent need to develop point-of-care detection methods to complement the gold standard laboratory-based PCR assay with comparable sensitivity and specificity. Herein, we developed a method termed mCAD to achieve ultraspecific point-of-care detection of SARS-CoV-2 RNA while maintaining high sensitivity by programming multiplex rolling circle amplification and toehold-mediated strand displacement reactions. RCA offers sufficient amplification of RNA targets for subsequent detection. Most importantly, a multilayer of detection specificity is implemented into mCAD via sequence-specific hybridization of nucleic acids across serial steps of this protocol to fully eliminate potential false-positive detections. Using mCAD, we demonstrated a highly specific, sensitive, and convenient visual detection of SARS-CoV-2 RNA from both synthetic and clinical samples, exhibiting performance comparable to qPCR. We envision that mCAD will find its broad applications in clinical prospects for nucleic acid detections readily beyond SARS-CoV-2 RNA.

A Colorimetric Biosensing Platform with Aptamers, Rolling Circle Amplification and Urease-Mediated Litmus Test.

Here we report on an ultra-sensitive colorimetric sensing platform that takes advantage of both the strong amplification power of rolling circle amplification (RCA) and the high efficiency of asimple urease-mediated litmus test.The presence of atarget triggers the RCA reaction, and urease-labelled DNA can hybridize to the biotinylated RCA products and beimmobilized onto streptavidin-coated magnetic beads. The urease-laden beads are then used to hydrolyze urea, leading to an increase in pH that can be detected by a simple litmus test. We show this sensing platform can be easily integrated with aptamers for sensing diverse targets via the detection of human thrombin and platelet-derived growth factor (PDGF) utilizing structure-switching aptamers as well as SARS-CoV-2 in human saliva using a spike-binding trimeric DNA aptamer. Furthermore, we demonstrate that this colorimetric sensing platform can be integrated into a simple paper-based device for sensing applications.