Colorimetric Readout Research Articles

Droplet Microarray as a Powerful Platform for Seeking New Antibiotics Against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria is a severe threat to public health. Therefore, it is urgent to establish effective screening systems for identifying novel antibacterial compounds. In this study, a highly miniaturized droplet microarray (DMA) based high-throughput screening system is established to screen over 2000 compounds for their antimicrobial properties against carbapenem-resistant Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus (MRSA). The DMA consists of an array of hydrophilic spots divided by superhydrophobic borders. Due to the differences in the surface wettability between the spots and the borders, arrays of hundreds of nanoliter-sized droplets containing bacteria and different drugs can be generated for screening applications. A simple colorimetric viability readout utilizing a conventional photo scanner is developed for fast single-step detection of the inhibitory effect of the compounds on bacterial growth on the whole array. Six hit compounds, including coumarins and structurally simplified estrogen analogs are identified in the primary screening and validated with minimum inhibition concentration assay for their antibacterial effect. This study demonstrates that the DMA-based high-throughput screening system enables the identification of potential antibiotics from novel synthetic compound libraries, offering opportunities for development of new treatments against multidrug-resistant bacteria.

A paper-based assay for the colorimetric detection of SARS-CoV-2 variants at single-nucleotide resolution.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for versatile diagnostic assays that can discriminate among emerging variants of the virus. Here we report the development and performance benchmarking of an inexpensive (approximately US$0.30 per test) assay for the rapid (sample-to-answer time within 30 min) colorimetric detection of SARS-CoV-2 variants. The assay, which we integrated into foldable paper strips, leverages nucleic acid strand-displacement reactions, the thermodynamic energy penalty associated with single-base-pair mismatches and the metal-ion-controlled enzymatic cleavage of urea to amplify the recognition of viral RNAs for the colorimetric readout of changes in pH via a smartphone. For 50 throat swab samples, the assay simultaneously detected the presence of SARS-CoV-2 and mutations specific to the SARS-CoV-2 variants Alpha, Beta and Gamma, with 100% concordance with real-time quantitative polymerase chain reaction and RNA sequencing. Customizable and inexpensive paper-based assays for the detection of viruses and their variants may facilitate viral surveillance.

Point-of-care non-invasive enzyme-cleavable nanosensors for acute transplant rejection detection

Accurate and non-invasive monitoring of allograft posttransplant is essential for early detection of acute cellular rejection and determines the long-term survival of the graft. Clinically, tissue biopsy is the most effective approach for diagnosing transplant rejection. Nonetheless, the procedure is invasive and potentially triggers organ failure. This work aims to design and apply GzmB-responsive nanosensors (GBRNs) that can readily size-change in graft tissues. Subsequently, we investigate the activity of serine protease granzyme B by generating a direct colorimetric urinary readout for non-invasive detection of transplant rejection in under 1 h. In preclinical heart graft mice models of transplant rejection, GBRNs were cleaved by GzmB and excreted by the kidneys via accurate nanometre-size glomerular filtration. By exploiting the catalytic activity of ultrasmall gold nanoclusters, GBRNs urinalysis promotes ultrasensitive surveillance of rejection episodes with a receiver operator characteristic curve area under the curve of 0.896 as well as a 95% confidence interval of about 0.7701–1.000. Besides, the catalytic activity of gold nanoclusters in urine can be detected at point-of-care testing to predict the immunity responses in mice with insufficient immunosuppressive therapy. Therefore, this non-invasive, sensitive, and quantitative method is a robust and informative approach for rapid and routine monitoring of transplant allografts without invasive biopsy.

Resettable sweat-powered wearable electrochromic biosensor

Wearable biofuel cells (BFCs) are being widely studied as self-powered sensors to monitor one's physiological status non-invasively. However, the energy generated by such devices is often insufficient to power accompanying measurement read-out systems and communication protocols. Here, we report a reversible fully printed electrochromic self-powered sensor (ESPS) toward on-body sweat lactate monitoring. The device was realized by integrating a lactate-oxidizable BFC anode, oxygen-reducible BFC cathode, and a reversible Prussian blue (PB) electrochromic display on a single wearable patch. Through modification of the BFC electrodes, the formal potential of the PB display (0.14 V) could be positioned in between the potentials of the lactate oxidation (−0.05 V) and oxygen reduction (0.35 V). Based on this system, the ESPS enables reversible and robust color changes with distinct time- and concentration-dependent profiles. The results demonstrate that the self-powered device has practical on-body applications for continual lactate monitoring with several advantages, including facile reversibility, ease of fabrication, and naked-eye colorimetric read-out.

Double Signal Amplification Strategy for Dual-Analyte Fluorescent Aptasensors for Visualizing Cancer Biomarker Proteins

The simultaneous analysis of diversified biomarkers with high sensitivity and in a point-of-care (POC) manner is of great significance for facile and early cancer diagnosis. Herein, we develop a target amplification-assisted ratiometric fluorescence assay (TARFA) platform integrating the dual-amplification strategy and colorimetric readout technology for sensitive and specific detection of two malignancy-associated biomarkers. Meanwhile, the NIR-excited alkaline-earth sulfide nanodots (ASNDs) with an ultrasmall (<10 nm) diameter and tunable emission wavelength are employed to replace commonly UV/visible light-excited fluorescent labels to minimize background interference from the sample matrix. Unique advantages of the ASNDs, together with superiority of consecutive signal amplification of enzymatic target recycling (ETR) and hybridization chain reaction (HCR), realize the pg/mL-range detection limit in specifically recognizing the vascular endothelial growth factor (VEGF) and soluble interleukin-6 receptors (sIL-6R). The combination detection of the dual analyte exhibits an improved sensitivity for cancer diagnosis. The addition of the target biomarkers leads to an increasingly ratiometric RGB signal, and quantification based on the ratio-dependent signal is more reliable rather than measuring the absolute RGB signals. Moreover, perceptible color transformation makes the TARFA platform competent for visual analysis of the target analytes as convenient as reading the pH indicator strip, and hue-based image analysis also improves the method with fine precision by quantitatively identifying the visual color. This work provides a new kind of NIR-excited aptasensing platform with a low detection limit, high throughput, and great portability, which also highlights the potential of the ASNDs in biomolecular fluorescent labeling.

(Digital Presentation) Stochastic Impact Electrochemistry in a Lateral-Flow Sensor Architecture

During the recent SARS-CoV-2 pandemic, lateral flow assays (LFAs) have written an indescribable story of success, as they provide means for decentralized, low-cost, and easy-to-use testing. However, LFAs in their most common form support only qualitative results and their sensitivity and limit of detection is significantly limited by the colorimetric readout method. In contrast, single-impact electrochemistry offers the possibility to quantify species beyond picomolar concentrations by recording individual species collisions with a biased microelectrode. Within this work, we investigate the integration of stochastic sensing into a LFA-architecture by combining a wax-patterned microchannel with a microelectrode array in order to detect silver nanoparticles by their oxidative dissolution. Here, we demonstrate the possibility to resolve individual nanoparticle collisions in a paper-based microchannel using a simplified reference-on-chip setup. Furthermore, we simulated a lateral-flow sensor, by flushing previously dried silver nanoparticles towards the electrode array, where the particles are subsequently detected. This proof-of-principle illustrates that single-impact electrochemistry might be a promising technique to extend the capability of LFAs. Especially, the integration of functionalized nanoparticle labels could enable the rapid and on-site detection of very dilute species with exceptional sensitivity.

M-CDC: Magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system.

The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M-CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M-CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments. In this paper, step-by-step instructions for M-CDC assay, including recombinase polymerase amplification (RPA)/reverse transcription-polymerase chain reaction (RT-RPA) of DNA or RNA, Cas12a-mediated target recognition and cleavage, and subsequent magnetic beads-mediated colorimetric readouts are provided. In addition, the protocol for the expression and purification of Lachnospiraceae bacterium-Cas12a (LbCas12a) protein, the design and synthesis of high-efficient crRNA, and the preparation of AuNP bioprobe are also offered.

Rapid Identification and Classification of Pathogens That Produce Carbapenemases and Cephalosporinases with a Colorimetric Paper-Based Multisensor.

Infections caused by bacteria that produce β-lactamases (BLs) are a major problem in hospital settings. The phenotypic detection of these bacterial strains requires culturing samples prior to analysis. This procedure may take up to 72 h, and therefore it cannot be used to guide the administration of the first antibiotic regimen. Here, we propose a multisensor for identifying pathogens bearing different types of β-lactamases above the infectious dose threshold within 90 min that does not require culturing samples. Instead, bacterial cells are preconcentrated in the cellulose scaffold of a paper-based multisensor. Then, 12 assays are performed in parallel to identify whether the pathogens produce carbapenemases and/or cephalosporinases, including metallo-β-lactamases, extended-spectrum β-lactamases (ESBLs), and AmpC enzymes. The multisensor generates an array of colored spots that can be quantified with image processing software and whose interpretation leads to the detection of the different enzymes depending on their specificity toward the hydrolysis of certain antibiotics, and/or their pattern of inhibition or cofactor activation. The test was validated for the diagnosis of urinary tract infections. The inexpensive paper platform along with the uncomplicated colorimetric readout makes the proposed prototypes promising for developing fully automated platforms for streamlined clinical diagnosis.

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/μL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

Enzyme-free ratiometric fluorescence and colorimetric dual read-out assay for glyphosate with ultrathin g-C3N4 nanosheets

Herein, a sensing platform for the rapid quantitative detection of glyphosate with high sensitivity and selectivity was constructed by enzyme-free ratiometric fluorescence and colorimetric dual read-out assay based on ultrathin g-C3N4 nanosheets (UCN NS).Fluorescent diaminophenylazine (DAP) with fluorescence was from the oxidation of o-phenylenediamine (OPD) in the presence of Cu2+. DAP could inspire a significant fluorescence quenching of nanosheets due to photo-induced electron transfer (PET), therefore exhibiting a ratiometric fluorescence response. In the presence of glyphosate, the strong chelation of Cu2+ by glyphosate inhibited the oxidation of OPD, which reduced the fluorescence intensity of DAP, while the fluorescence of the ultrathin nanosheets was restored. Moreover, the oxidation of OPD (colorless) to DAP (yellow) was a distinctly visible color change process, which provides well-suited conditions for colorimetric analysis. At the optimum experimental conditions, the detection concentration range of glyphosate was 0.001–0.48 μg/mL, and the limit of fluorometric detection was 0.49 ng/mL (S/N = 3). This assay was simple and easy to use for the detection of glyphosate in food safety and the environment with satisfactory results.

Dibromorhodamine‐based photoredox catalysis under visible light for the colorimetric detection of Hg(II) ion

AbstractWe demonstrated a colorimetric readout of Hg(II) ion through visible‐light‐induced polymerization, initiated by an Hg(II)‐responsive photoredox initiator/catalyst under aerobic conditions. Thiosemicarbazide‐functionalized 4′,5′‐dibromorhodamine was synthesized as a stimuli‐responsive photoredox catalyst. The rhodamine derivatives bearing thiosemicarbazide have been known as fluorogenic probes of Hg(II) ion with excellent sensitivity and selectivity. In this work, we incorporated a heavy halogen atom (i.e., Br) onto the xanthene core ring of the rhodamine derivative to improve the photoredox catalytic performance of rhodamine 6G, which exhibits a low catalytic performance, presumably due to the poor quantum yield of intersystem crossing. Thus, this photoredox catalyst led to the formation of a hydrogel with Hg(II) ion in a concentration of 10 μM, as the catalyst initiated free‐radical polymerization of poly(ethylene glycol) diacrylate and N‐vinylpyrrolidone with triethanolamine under visible light for 1 min.

Enzyme-free dual-amplification assay for colorimetric detection of tetracycline based on Mg2+-dependent DNAzyme assisted catalytic hairpin assembly.

Detection of ultralow concentration of tetracycline (TC) plays a key role in food safety and human health. Herein, we fabricated an enzyme-free dual-amplification assay for sensitive detection of TC in milk. The sensing system ingeniously combined Mg2+-dependent DNAzyme (MNAzyme) cleavage and catalytic hairpin assembly (CHA). Through the binding of TC and specific aptamer (Apt), DNA1 was released from the Apt-DNA1 complex. Then the separated DNA1 would hybridize with DNA2 to activate the catalytic activity of MNAzyme, which subsequently cleaved the substrate H0 and generated a new unit to trigger the following CHA reaction between H1 and H2, thereby releasing the G-rich sequence of H1 to induce the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. Under optimal conditions, the absorption intensity exhibited a significant linear correlation with the logarithm of the target TC concentration over a range of 4 orders of magnitude dynamic range from 1 pM to 10nM. The limit of detection was calculated to be 0.89 pM and the method showed high selectivity for TC. More impressively, the proposed method was employed for TC determination in the milk sample.

Compact Microfluidic Platform with LED Light-Actuated Valves for Enzyme-Linked Immunosorbent Assay Automation.

Lab-on-a-chip devices incorporating valves and pumps can perform complex assays involving multiple reagents. However, the instruments used to drive these chips are complex and bulky. In this article, a new wax valve design that uses light from a light emitting diode (LED) for both opening and closing is reported. The valves and a pumping chamber are integrated in lab-on-a-foil chips that can be fabricated at low cost using rapid prototyping techniques. A chip for the implementation of enzyme-linked immunosorbent assays (ELISA) is designed. A porous nitrocellulose material is used for the immobilization of capture antibodies in the microchannel. A compact generic instrument with an array of 64 LEDs, a linear actuator to drive the pumping chamber, and absorbance detection for a colorimetric readout of the assay is also presented. Characterization of all the components and functionalities of the platform and the designed chip demonstrate their potential for assay automation.

Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.

Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.

Simultaneous detection of multiple foodborne bacteria by loop-mediated isothermal amplification on a microfluidic chip through colorimetric and fluorescent assay

The integration of loop-mediated isothermal amplification (LAMP) onto a microfluidic chip adds more simplicity and portability for food safety control. However, this system for multiple-target detection was less used on foodborne bacteria. Here, we utilized a ten-well microfluidic chip with pre-loaded LAMP primer sets for the quantitative and qualitative detection of Salmonella , Staphylococcus aureus , Escherichia coli O157:H7, and Shigella . The specificity and sensitivity of designed primers targeting sirA , spA , fliC , and ipaH genes were validated. Using regular tubes and the ten-well microfluidic chips, these four pathogens could be simultaneously detected within 45 min by LAMP with fluorescent and colorimetric readouts, respectively. Finally, the results demonstrated that the limit of detection reached 8 × 10 3 CFU mL −1 genomic DNA with a recovery rate of 86.1%∼110%. In summary, our microfluidic chip platform displayed obvious advantages in time-saving, cost-effectiveness, and sensitivity, which is applicable for in-field monitoring of food pathogens. ● LAMP reaction is integrated on a 10-well microfluidic chip for food detection. ● The microfluidic chip is used for both quantitative and qualitative detection. ● The fluorescent intensity and colorimetric amplification readouts can be obtained. ● This microfluidic chip platform simultaneously detects four bacteria within 45 min.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

A point-of-use lateral flow aptasensor for naked-eye detection of aflatoxin B1

Aflatoxin B1 (AFB1) contamination in food is not only a serious health threat to humans, but also a considerable economic burden worldwide. Rapid and accurate detection of AFB1 during production, processing, transportation and storage is therefore a prerequisite to prevent spreading of AFB1. Here, we developed a simple and easy-to-use lateral flow aptasensor (LFA) to visually detect AFB1 using AFB1 aptamer. Polystyrene dyed particles were used as a signal reporter to enable colorimetric readout by naked eyes, in which the signal is directly proportional to the concentration of AFB1. Both aptamer specificity and target induced strand release approach enable a single-step AFB1 detection. The whole assay took 35 min to complete, starting from sample/dye complex incubation to color acquisition. Under the optimized condition, this sensing platform could detect AFB1 as low as 4.56 ng mL−1 with two distinct linear ranges of 0–0.05 μg mL−1 and 0.05–1 μg mL−1. LFA validation with 4 spiked samples (peanut, corn, rice and chili powder) showed satisfying AFB1 recovery within the range of 90.83–110.21%. Our LFA platform could potentially be applied to the on-site, naked-eye, and instrument-free detection of AFB1 or other substances with available aptamers.

Portable silver-doped prussian blue nanoparticle hydrogels for colorimetric and photothermal monitoring of shrimp and fish freshness

Herein, we explored a colorimetric and photothermal readout strategy based on silver-doped prussian blue nanoparticles (SPB NPs) hydrogel for detection of trimethylamine (TMA) and monitoring of shrimp/fish freshness. The functional hydrogel is fabricated via the addition of SPB NPs to agarose hydrogel. TMA vapor can permeate into the functional hydrogel, thereby causing the decomposition of SPB NPs along with the color evolution (blue to colorless) and the disappearance of photothermal effects. The color information was digitized by a smartphone combined with RGB (red/green/blue) analysis. The photothermal effects was recorded by a handheld thermal imager. The ∆R gray value and temperature evolutions of the functional hydrogel were all closely related to the concentration of TMA, making it successful in the assessment of shrimp/fish freshness. Both colorimetric and photothermal signals are linear response to TMA concentration from 0.21 to 0.54 ppm. The application of smartphone and handheld thermal imager greatly improves the portability and practicality of monitoring on-spot. More significantly, the photothermal signal can still be applied to assess food freshness, when the hydrogel is contaminated by the colored food substrates and the colorimetric signal cannot be used to assessment. To our knowledge, this is the first time that a photothermal strategy has been developed to monitor food freshness. Therefore, this simple, cost-effective, nondestructive and sensitive dual-mode functional hydrogel will open up a new prospect for monitoring of food spoilage.

Reversible photonic hydrogel sensors via holographic interference lithography

Continuous monitoring of physiological conditions and biomarkers via optical holographic sensors is an area of growing interest to facilitate the expansion of personalised medicine. Here, a facile laser-induced dual polymerization method is developed to fabricate holographic hydrogel sensors for the continuous and reversible colorimetric determination of pH variations over a physiological range in serum (pH 7–9). Readout parameters simulated through a Finite-difference time-domain Yee's algorithm retrieve the spectral response through expansion. Laser lithography of holographic hydrogel sensor fabrication is achieved via a single 355 nm laser pulse to initiate polymerization of ultrafine hydrogel fringes. Eliminating the requirement for complex processing of toxic components and streamlining the synthetic procedure provides a simpler route to mass production. Optimised pH-responsive hydrogels contain amine bearing functional co-monomers demonstrating reversible Bragg wavelength shifts of 172 nm across the entire visible wavelength range with pH variation from 7.0 to 9.0 upon illumination with broadband light. Photolithographic recording of information shows the ability to convey detailed information to users for qualitative identification of pH. Holographic sensor reversibility over 20 cycles showed minimal variation in replay wavelength supporting reliable and consistent readout, with optimised sensors showing rapid response times of <5 min. The developed sensors demonstrate the application to continuous monitoring in biological fluids, withstanding interference from electrolytes, saccharides, and proteins colorimetrically identifying bovine serum pH over a physiological range. The holographic sensors benefit point-of-care pH analysis of biological analytes which could be applied to the identification of blood gas disorders and wound regeneration monitoring through colorimetric readouts.

One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.