Visual Readout Research Articles

Portable self-powered electrochemical aptasensor for ultrasensitive and real-time detection of microcystin-RR based on hydrovoltaic-photothermal coupling effect

Coupling different energy harvesting technologies to obtain an excellent output signal is essential for the development of high-performance self-powered electrochemical sensors. Herein, a novel hydrovoltaic-photothermal coupling self-powered electrochemical aptasensing platform was designed for sensitive detection of microcystin (MC-RR) with a digital multimeter as a direct visual readout strategy. The straightforward ultrasonic method was employed to synthesize polyaniline (PANI) and bismuth oxybromide (BiOBr) nanosheets, which were then integrated as active components in a hydrovoltaic device. The unique layer structure of two-dimensional (2D) nanomaterials BiOBr can create flexible interlayer spaces to accommodate various ions and water molecules, which was beneficial to construct evaporation-driven channels. Meanwhile, the exceptional photothermal characteristics of polyaniline could accelerate the water evaporation rate, consequently boosting the migration speed of charge carriers and increasing output signal. Moreover, a digital multimeter was connected to the constructed sensor for real-time displaying the output signal. With the assistance of aptamer, a novel self-powered electrochemical aptasensing platform was constructed for sensitive detection of MC-RR. Under optimum conditions, the output signal of the hydrovoltaic-photothermal coupling cell was linearly related to the logarithm of MC-RR concentration in the range of 1 fM to 1 nM with a detection limit of 0.31 fM (S/N = 3). Furthermore, this sensor also exhibited many advantages such as high selectivity, good repeatability and portability. Such novel strategy not only offers a completely new general approach to construct high-performance self-powered devices for the detection of MC-RR, but also provides a new strategy for advancing the miniaturization and field application of self-powered electrochemical sensors.

A-327 Statistical Technique-based Image Analysis for Digital Reading of Immunochemistry Cassette

Abstract Background Covid-19 transformed healthcare and how we think about clinical diagnostics. People prefer at home testing options that enable them to focus on staying healthy at their own comfort and privacy. Fast and accurate lateral flow assays or rapid diagnostic test (RDT) meets that need, however ensuring high test specificity and accuracy are of utmost importance. Visual readout of RDTs is subjective and time consuming. With the current technological advancement, medical devices are smarter and can be used for automated readout. The core of such devices are the algorithms that process the information despite several limitations and unknown variations affecting input signals. Many such algorithms are developed and are used for similar applications. Here, a statistical model for any optical device that is designed to automatically read an RDT with one or more test lines is described. Methods The described LFA algorithm is a innovative way to analyze images acquired with Complementary Metal-Oxide-Semiconductor (CMOS) based optical system with low Signal-to-noise ratio (SNR), giving a semi quantitative result. This also takes care of variations in illumination sources with relatively reduced computation, by converting the images to a 1 Dimensional (1D) signal. Algorithm workflow: Image pre-processing: Affine transformations such as rotation, translation, etc. (depends on the inherent setup) are applied on the acquired images as a pre-processing step. Pattern matching techniques using Gaussian templates are applied to determine the window region of the LFA and precisely identify the test and control line regions. Image formatting: The sample region of the cassette is converted from RGB color space to the weighted gray (Gw) for digital interpretation of the primary biomarker present. Characterization of the Gw channel enables test peak detection with enhanced resolution. This helps in early call out of results and improved accuracy, thereby reducing chances of erroneous results. A second order Butterworth low pass filter is applied to reduce system noise and enhance SNR. The images are converted to 1D signal by averaging for mathematical read out. Mathematical interpretation: Maximum peak height (max) and minimum peak depth (min) of Gw channel values in the test line region (t-peak) are extracted from the 1D signal. The mathematical representation of the test line is calculated as: Test Peak Signal = {max(t-peak) - min(t-peak)} / Alpha; Normalization Factor, Alpha = (sum of all Gw channel values for any clear non-reactive part of the test device) / (number of Gw channel values) Results TPS values for various biomarkers from statistically significant replicates were computed at Low, Medium, and High levels. Two sample T-tests were performed, and respective p values are distinctly different as p-value<0.05. For a concentration level of Low to medium, p-value is 0.044 and for Medium - High p-value is 0.004. Conclusions This algorithm can run on any off-the-shelf available RDT coupled with a CMOS based optical system. The proposed algorithm can differentiate between low, medium, and high concentrations with high confidence. High throughput semiquantitative digital readouts are robust, accurate and are preferred for diagnostic test result interpretation.

Direct comparison of colorimetric signal amplification techniques in lateral flow immunoassays.

The lateral flow immunoassay (LFIA) is widely adopted for point-of-care testing, but its limit of detection (LoD) falls short of that of laboratory-based immunoassays. Several techniques have been proposed to enhance the LoD of LFIAs using visual colorimetric readouts, yet a direct comparison of the LoDs achieved by these techniques has not been performed. In this work, we measure the LoDs of LFIAs designed for the detection of a malaria protein, PfHRP2, using four different colorimetric signal generation techniques: (i) AuNP(40 nm)-tagged detection antibodies (base case), (ii) AuNP-based enhancement of AuNP(40 nm) signal, (iii) oxidation of chloronaphthol/diaminobenzidine (CN/DAB) using HRP-tagged detection antibodies, and (iv) oxidation of CN/DAB using polyHRP(400)-tagged detection antibodies. The LoDs and the 95% confidence intervals of the LoDs achieved by the 4 techniques were 19.34 (13.37-27.62) ng mL-1, 9.57 (6.76-13.28) ng mL-1, 21.57 (14.26-32.18) ng mL-1, and 6.09 (2.23-13.47) ng mL-1, respectively. Contrary to popular perception, enzymatic signal generation using HRP-tagged detection antibodies did not improve the LoD compared to the base case of AuNP-based signal generation. Further studies revealed that the very high extinction coefficient of gold nanoparticles renders them an excellent choice for colorimetric detection, surpassing the performance of enzymatic signal generation using HRP-tagged antibodies. However, enzymatic signal generation using polyHRP-tagged antibodies improved the LoD compared to the base case. These results show that enzymatic signal amplification should not be a priori assumed to be superior to AuNP-based signal generation; and provide a reference point to LFIA developers to select an appropriate signal generation modality.

One-Tube Nested PCR Coupled with CRISPR-Cas12a for Ultrasensitive Nucleic Acid Testing.

Nucleic acid testing with high sensitivity and specificity is of great importance for accurate disease diagnostics. Here, we developed an in situ one-tube nucleic acid testing assay. In this assay, the target nucleic acid is captured using magnetic silica beads, avoiding an elution step, followed directly by the polymerase chain reaction (PCR) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a detection. This assay achieved visual readout and a sensitivity of 120 copies/mL for detecting SARS-CoV-2. More importantly, the assay demonstrated over 95% sensitivity and 100% specificity compared to the gold standard real-time quantitative PCR (RT-qPCR) test by using 75 SARS-CoV-2 clinical samples. By integrating nested PCR and Cas12a, this all-in-one nucleic acid testing approach enables ultrasensitive, highly specific, and cost-effective diagnosis at community clinics and township hospitals.

AuNP@Self-assembled 4H-Chromene-Enhanced colorimetric detection of ganciclovir using smartphone Technology: Portable solutions for healthcare

Any substance, radiation, or radionuclide in our environment that promotes carcinogenesis is a cancerogen. In the current work, we modified the 4H-chromene derivative by covering its surface with a shell of gold nanoparticles. Interestingly, ganciclovir (GCV) formed a bridge between the Nap-group of 4H-chromene and gold nanoparticles to display surface charge reduction properties in AuNP@Self-assembled 4H-Chromene. The surface charge of AuNPs is reduced as a result of the electron transfer mechanism brought on by the creation of the Nap-GCV-AuNP link, which leads to agglomeration. This led to the development of a dual-mode colorimetric sensor for GCV that incorporates solution form and portable sensor strip. The probe showed a linear range of 0–74 nM under ideal circumstances, with a detection limit of 4 nM in solution and 3 nM on paper strip detection. An evaluation of the practical utility in human serum using a portable visual read-out produced satisfactory findings, with a recovery percentage of 102.1 % and a standard deviation of ± 4 %. Surprisingly, this is the first work on the colorimetric detection of Ganciclovir that we are aware of.

Metabolic engineering-enabled dual-color biosensor for discriminative and sensitive detection of toxic lead and mercury in environmental waters

The ubiquity of toxic heavy metals, particularly lead (Pb) and mercury (Hg), in environmental waters severely threatens ecosystems and human health. We developed a metabolic engineering-based dual-color bacterial biosensor for the sensitive and discriminative detection of Pb(II) and Hg(II) at nanomolar levels. The biosensor leverages the synthetic biology approach to integrate two pigments, prodeoxyviolacein (PDV) and deoxyviolacein (DV), into a single construct that responds distinctly to Pb(II) and Hg(II), respectively. The engineered E. coli biosensor exhibits a detection range of 0.732–3000 nM for Pb(II) and 0.183–750 nM for Hg(II), with low detection limits of 0.732 nM for Pb(II) and 0.183 nM for Hg(II). The biosensor demonstrated high sensitivity, selectivity, and accuracy with a recovery rate close to 1 in freshwater and seawater matrices. Notably, the biosensor's visual readout allows for rapid, equipment-free assessment. It is a practical tool for real-world monitoring of heavy metal pollution, especially in remote or resource-limited settings. This study underscores the potential of whole-cell biosensors as a sustainable and effective alternative to traditional heavy metal detection methods.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

Enhancing efficiency in detection of COVID-19 through AI-driven colorimetric isothermal detection with multiplex primers

COVID-19 has afflicted millions of lives worldwide. Although there are many rapid methods to detect it based on colorimetric loop-mediated isothermal amplification, there remains room for improvement. This study aims to 1) integrate multiple primers into a singleplex assay to enhance the diagnostic sensitivity, and 2) utilize a high-throughput smartphone-operatable AI-driven color reading tool to enable a rapid result analysis. This setup can improve the sensitivity by 10–100 times and can analyze approximately 6700 samples per minute. The assay is simpler than RT-qPCR, with a turnaround time of less than 75 min. It can detect various types of SARS-CoV-2 by targeting 3 genes, increasing the likelihood that it will remain effective even if the virus undergoes mutations in any single target gene. In summary, it affords potential for adaptation to detection of new/re-emerging diseases with the visual readout for maximum assay simplicity and AI-operated mode for large-scale testing.

Diffusion-driven height readout analytical device based on gold nanobipyramid-doped supramolecular hydrogel for point-of-care bioanalysis

Point-of-care (POC) diagnostics is crucial for rapid on-site detection in resource-limited settings. However, there are still some challenges in the development of quantitative POC bioassays. Herein, we propose a simple, visual, and instrument-free height readout analytical device injected with target-responsive gold nanobipyramid (AuNBP)-doped guanosine-quartet (G4) supramolecular hydrogel for the quantitative detection of glucose. This sensing platform relies on H2O2 generated by glucose oxidation to induce iodide-mediated etching of AuNBPs, resulting in visualized color-changed hydrogel bar-chart driven by molecular diffusion in a polycarbonate tube. The visual height of the color-changed hydrogel is correlated with the concentration of glucose, and the quantitative determination was achieved simply by using a household ruler or mobile software as a readout. More importantly, this method has been successfully used to detect glucose in human serum samples with high accuracy and reliability. In addition, the AuNBPs are utilized as plasmonic probes to construct two-input logic gates, providing an intelligent strategy to simultaneously detect multiple biomolecules. This height-readout sensing platform demonstrates great promise for applications in POC and intelligent bioanalysis.

Digitalization of Colorimetric Sensor Technologies for Food Safety.

Colorimetric sensors play a crucial role in promoting on-site testing, enabling the detection and/or quantification of various analytes based on changes in color. These sensors offer several advantages, such as simplicity, cost-effectiveness, and visual readouts, making them suitable for a wide range of applications, including food safety and monitoring. A critical component in portable colorimetric sensors involves their integration with color models for effective analysis and interpretation of output signals. The most commonly used models include CIELAB (Commission Internationale de l'Eclairage), RGB (Red, Green, Blue), and HSV (Hue, Saturation, Value). This review outlines the use of color models via digitalization in sensing applications within the food safety and monitoring field. Additionally, challenges, future directions, and considerations are discussed, highlighting a significant gap in integrating a comparative analysis toward determining the color model that results in the highest sensor performance. The aim of this review is to underline the potential of this integration in mitigating the global impact of food spoilage and contamination on health and the economy, proposing a multidisciplinary approach to harness the full capabilities of colorimetric sensors in ensuring food safety.

Enhancing Sensitivity in SARS-CoV-2 Rapid Antigen Testing through Integration of a Water-Soluble Polymer Wall.

Lateral flow immunoassays (LFIAs) are recognized for their practicality in homecare and point-of-care testing, owing to their simplicity, cost-efficiency, and rapid visual readouts. Despite these advantages, LFIAs typically fall short in sensitivity, particularly in detecting viruses such as SARS-CoV-2, thus limiting their broader application. In response to this challenge, we have innovated an approach to substantially enhance LFIA sensitivity. This involves the integration of a water-soluble dextran-methacrylate polymer wall with a 15% grafting degree positioned between the test and control lines on the LFIA strip. This novel modification significantly improved the sensitivity of the assay, achieving detection limits as low as 50 pg mL-1 and enhancing the sensitivity by 5-20-fold relative to existing LFIA kits available on the market. Furthermore, our developed LFIA kit (WSPW-LFIA) demonstrated exceptional specificity for SARS-CoV-2. Coupled with a straightforward fabrication process and robust stability, the WSPW-LFIA represents a promising advancement for real-time in vitro diagnosis across a spectrum of diseases.

Advanced Enzyme Mimicking Engineering: 3D Biomimetic Pt Single‐Atom Nanozymes Initiating Pressure‐Driven Device

AbstractCurrent research on single‐atom nanozymes has predominantly concentrated on 2D structures, with limited exploration into the influence of 3D biomimetic structures on their catalytic performance. In this work, a 3D nanostructure of Pt single‐atom nanozyme (Pt‐NC SAzyme) is fabricated by encapsulating platinum 2,4‐pentanedionate in each molecular cage of ZIF‐8 and then pyrolyzing. X‐ray absorption fine structure analysis confirmed the presence of Pt as single atoms, with each Pt atom exclusively coordinated with four nitrogen atoms, while the *O is identified as an intermediate in the catalytic reaction. Additionally, theoretical calculations demonstrated that Pt‐NC SAzyme exhibited a lower potential energy and a shorter pathway for catalyzing the production of O2 from H2O2 than that of 2D Pt‐NC due to the localization effect, confirming a superior catalase‐like activity of Pt‐NC SAzyme. Based on the efficient O2 production from Pt‐NC SAzyme, a dual‐mode pressure immunosensor is established to achieve sensitive detection of aminopyrine by converting gas‐induced pressure into visual and timing readouts. This work introduced a novel approach to enhance the catalytic activity of SAzymes through bionic structural design and coordination environment, while also proposing a new concept for point‐of‐care testing in food safety, environmental monitoring, and disease diagnosis.

Photocatalytic colorimetry and fluorescence dual-signal biosensor for instant determination of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia. Herein, a photocatalytic colorimetry and fluorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity. Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery.

Membrane-Free Lateral Flow Assay with the Active Control of Fluid Transport for Ultrasensitive Cardiac Biomarker Detection.

Membrane-based lateral flow immunoassays (LFAs) have been employed as early point-of-care (POC) testing tools in clinical settings. However, the varying membrane properties, uncontrollable sample transport in LFAs, visual readout, and required large sample volumes have been major limiting factors in realizing needed sensitivity and desirable precise quantification. Addressing these challenges, we designed a membrane-free system in which the desirable three-dimensional (3D) structure of the detection zone is imitated and used a small pump for fluid flow and fluorescence as readout, all the while maintaining a one-step assay protocol. A hydrogel-like protein-polyelectrolyte complex (PPC) within a polyelectrolyte multilayer (PEM) was developed as the test line by complexing polystreptavidin (pSA) with poly(diallyldimethylammonium chloride) (PDDA), which in turn was layered with poly(acrylic acid) (PAA) resulting in a superior 3D streptavidin-rich test line. Since the remainder of the microchannel remains material-free, good flow control is achieved, and with the total volume of 20 μL, 7.5-fold smaller sample volumes can be used in comparison to conventional LFAs. High sensitivity with desirable reproducibility and a 20 min total assay time were achieved for the detection of NT-proBNP in plasma with a dynamic range of 60-9000 pg·mL-1 and a limit of detection of 56 pg·mL-1 using probe antibody-modified fluorescence nanoparticles. While instrument-free visual detection is no longer possible, the developed lateral flow channel platform has the potential to dramatically expand the LFA applicability, as it overcomes the limitations of membrane-based immunoassays, ultimately improving the accuracy and reducing the sample volume so that finger-prick analyses can easily be done in a one-step assay for analytes present at very low concentrations.

Development of a Rapid Detection Method to Prorocentrum lima by Loop-Mediated Isothermal Amplification with Hydroxy Naphthol Blue.

Prorocentrum lima, a widely distributed dinoflagellate known for its production of harmful biotoxins, poses a significant threat to humans, aquaculture, and marine ecosystems. As a result, the detection of this toxic alga in coastal waters has become an urgent research focus. In this study, a rapid, sensitive, and cost-effective detection method based on loop-mediated isothermal amplification (LAMP) was developed to identify P. lima. In this method, cell extracts of P. lima were diluted and used directly as templates for amplification, eliminating the need for nucleic acid purification and simplifying the detection process. Hydroxy naphthol blue (HNB) was incorporated into the reaction mix to facilitate result interpretation, enabling visual determination of the amplification outcome with the naked eye. The entire detection process, from DNA extraction to template amplification and product detection, could be completed within 80min using a simple constant temperature-control device. This LAMP-based detection method demonstrated excellent reliability, specificity, and a low detection limit of 5.87 cells/mL for DNA crude extract. The assay offered an efficient alternative to PCR for rapid detection of P. lima. By streamlining the detection process and offering a visual readout, this technique holds promise for efficient and routine monitoring of harmful algal species, benefitting both research efforts and environmental management strategies.

Controllable detection threshold achieved through the toehold switch system in a mercury ion whole-cell biosensor

Due to the toxicity of mercury and its harmful effects on human health, it is essential to establish a low-cost, highly sensitive and highly specific monitoring method with a wide detection range, ideally with a simple visual readout. In this study, a whole-cell biosensor with adjustable detection limits was developed for the detection of mercury ions in water samples, allowing controllable threshold detection with an expanded detection range. Gene circuits were constructed by combining the toehold switch system with lactose operon, mercury-ion-specific operon, and inducible red fluorescent protein gene. Using MATLAB for design and selection, a total of eleven dual-input single-output sensing logic circuits were obtained based on the basic logic of gene circuit construction. Then, biosensor DTS-3 was selected based on its fluorescence response at different isopropyl β-D-Thiogalactoside (IPTG) concentrations, exhibiting the controllable detection threshold. At 5–20 μM IPTG, DTS-3 can achieve variable threshold detection in the range of 0.005–0.0075, 0.06–0.08, 1–2, and 4–6 μM mercury ion concentrations, respectively. Specificity experiments demonstrated that DTS-3 exhibits good specificity, not showing fluorescence response changes compared with other metal ions. Furthermore spiked sample experiments demonstrated its good resistance to interference, allowing it to distinguish mercury ion concentrations as low as 7.5 nM by the naked eye and 5 nM using a microplate reader. This study confirms the feasibility and performance of biosensor with controllable detection threshold, providing a new detection method and new ideas for expanding the detection range of biosensors while ensuring rapid and convenient measurements without compromising sensitivity.

Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.

Smart Free-Standing Bilayer Polyacrylamide/DNA Hybrid Hydrogel Film-Based Sensing System Using Changes in Bending Angles as a Visual Signal Readout.

Stimuli-responsive DNA hydrogels have shown great potential in sensing applications due to their attractive properties such as programmable target responsiveness, excellent biocompatibility, and biodegradability. In contrast to the extensively developed DNA hydrogel sensing systems based on the stimuli-responsive hydrogel-to-solution phase transition of the hydrogel matrix, the quantitative sensing application of DNA hydrogels exhibiting smart shape deformations has rarely been explored. Moreover, bulk DNA hydrogel-based sensing systems also suffer from high material cost and slow response. Herein, free-standing bilayer polyacrylamide/DNA hybrid hydrogel films with programmable responsive properties directed by the sequence of functional DNA units have been constructed. Compared with bulk DNA hydrogels, these DNA hydrogel films with a thickness at the micrometer scale not only greatly reduce the consumption of DNA materials but also facilitate the mass transfer of biomacromolecular substances within the hydrogel network, thus favoring their sensing applications. Therefore, a target-responsive smart DNA hydrogel film-based sensor system is further demonstrated based on the large amplitude macroscopic shape deformation of the film as a visual signal readout. As a proof of concept, Pb2+ or UO22+ ion-responsive DNA units were introduced into the active layer of the bilayer hydrogel films. In the presence of Pb2+ or UO22+ ions, the occurrence of a cleavage reaction within the DNA units leads to the release of DNA segments from the hydrogel film, inducing a dramatic shape deformation of the film, and thus sensing of Pb2+ or UO22+ ions with high specificity is achieved based on measuring the bending angle changes of these smart free-standing films. These smart DNA hydrogel film sensors with target-programmable responsiveness, simple operation, and ease of storage may hold promise for future rapid on-site testing applications.

Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay

BackgroundFeline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay.ResultsThe recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10− 1 copies/μL for the FHV-1 TK gene and 5.5 copies/μL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings.ConclusionsThe one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.

Integrating Paper-Based Microfluidics and Lateral Flow Strip into Nucleic Acid Amplification Device toward Rapid, Low-Cost, and Visual Diagnosis of Multiple Mycobacteria.

Efficient diagnosis of mycobacterial infections can effectively manage and prevent the transmission of infectiousdiseases. Unfortunately, existing diagnostic strategies are challenged by long assay times, high costs, and highly specialized expertise to distinguish between pulmonary tuberculosis (PTB) and nontuberculous mycobacterial pulmonary diseases (NTM-PDs). Herein, in this study, an optimized 3D paper-based analytical device (µPAD) is incorporated with a closed lateral flow (LF) strip into a loop-mediated isothermal amplification (LAMP)device (3D-µPAD-LF-LAMP) for rapid, low-cost, and visual detection of pathogenic mycobacteria. The platform's microfluidic featureenhanced the nucleic acid amplification, thereby reducing the costs and time as compared to boiling, easyMAG, and QIAGEN techniques. Moreover, the LF unitis specifically designed to minimize aerosol contamination for a user-friendly and visual readout. 3D-µPAD-LF-LAMP is optimized and assessed using standard strains, demonstrating a limit of detection (LOD) down to 10 fgreaction-1 . In a cohort of 815 patients, 3D-µPAD-LF-LAMP displays significantly better sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and diagnostic accuracy than conventional bacterial culture and Xpert techniques. Collectively, 3D-µPAD-LF-LAMP demonstrates enhanced accessibility, efficiency, and practicality for the diagnosis of multiple pathogenic mycobacteria, which can be applied across diverse clinical settings, thereby ultimately improving public health outcomes.