Unveiling Cas12j Trans-Cleavage Activity for CRISPR Diagnostics: Application to miRNA Detection in Lung Cancer Diagnosis.

Cas12j, a hypercompact and efficient Cas protein, has potential for use in CRISPR diagnostics, but has not yet been used because the trans-cleavage activity of Cas12j is veiled. Here, the trans-cleavage behavior of Cas12j1, 2, and 3 variants and evaluate their suitability for nucleic acid detection is unveiled. The target preferences and mismatch specificities of the Cas12j variants are precisely investigated and the optimal Cas12j reaction conditions are determined. As a result, the EXP-J assay for miRNA detection by harnessing the robust trans-cleavage activity of Cas12j on short ssDNA is developed. The EXP-J method demonstrates exceptional detection capabilities for miRNAs, proving that Cas12j can be a pivotal component in molecular diagnostics. Furthermore, the translational potential of the EXP-J assay is validated by detecting oncogenic miRNAs in plasma samples from lung cancer patients. This investigation not only elucidates the trans-cleavage characteristics of Cas12j variants, but also advances the Cas12j-based diagnostic toolkit.

Small Molecule Detection with Ligation-Dependent Light-Up Aptamer Transcriptional Amplification.

ATP and NAD+ are small biomolecules that participate in a variety of physiological functions and are considered as potential biomarkers for disease diagnosis. In this study, we developed a ligation-dependent light-up aptamer transcriptional amplification assay for the sensitive and selective detection of ATP and NAD+. This assay relies on a specific DNA ligase that catalyzes the ligation of a nicked DNA template in the presence of a specific small molecule. We prepared a nicked template consisting of a duplex fragment with an overhang for the T7 promoter region and a single-stranded DNA with a complementary overhang sequence for the Broccoli aptamer. The nicked template was connected using a DNA ligase in the presence of a specific small molecule. The ligation product was subjected to in vitro transcription to amplify the light-up aptamer-mediated fluorescence signals. By integrating the target-dependent ligation and transcription amplification, significant signal amplification was achieved with 5.9 and 142 pM detection limits for ATP and NAD+, respectively. Moreover, good selectivity to discriminate between the target and its analogues was also realized. The application of this method to biological samples was evaluated using human serum and exhibited excellent recovery values.

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.

MicroRNA detection using light-up aptamer amplification based on nuclease protection transcription.

The quantification of microRNAs (miRNAs) is important because the miRNA expression level is closely associated with the occurrence and development of diseases. Here, we report a simple nuclease protection transcription assay which combines nuclease protection assays and transcription-assisted light-up aptamer amplification for detecting miRNAs with great sensitivity.

Nanoplasmonic immunosensor for the detection of SCG2, a candidate serum biomarker for the early diagnosis of neurodevelopmental disorder.

The neural circuits of the infant brain are rapidly established near 6 months of age, but neurodevelopmental disorders can be diagnosed only at the age of 2-3 years using existing diagnostic methods. Early diagnosis is very important to alleviate life-long disability in patients through appropriate early intervention, and it is imperative to develop new diagnostic methods for early detection of neurodevelopmental disorders. We examined the serum level of secretogranin II (SCG2) in pediatric patients to evaluate its potential role as a biomarker for neurodevelopmental disorders. A plasmonic immunosensor performing an enzyme-linked immunosorbent assay (ELISA) on a gold nanodot array was developed to detect SCG2 in small volumes of serum. This nanoplasmonic immunosensor combined with tyramide signal amplification was highly sensitive to detect SCG2 in only 5 µL serum samples. The analysis using the nanoplasmonic immunosensor revealed higher serum SCG2 levels in pediatric patients with developmental delay than in the control group. Overexpression or knockdown of SCG2 in hippocampal neurons significantly attenuated dendritic arborization and synaptic formation. These results suggest that dysregulated SCG2 expression impairs neural development. In conclusion, we developed a highly sensitive nanoplasmonic immunosensor to detect serum SCG2, a candidate biomarker for the early diagnosis of neurodevelopmental disorders.

Aptamer-linked in vitro expression assay for ultrasensitive detection of biomarkers.

Signal amplification is a key step that determines the sensitivity of molecular assays. Although studies on aptamers have mostly focused on their target-binding ability, taking advantage of the gene-coding function of nucleic acids, we demonstrate here that aptamers can be engineered into diagnostic reagents that can both recognize a target and generate highly amplified detection signals. We developed a strategy that employs a 'readable' aptamer that consists of a single-stranded aptamer and a double-stranded reporter gene. After binding to its target via the aptamer region, the reporter gene of the readable aptamer produces amplified number of signal-generating enzymes through a subsequent in vitro expression reaction. In contrast to conventional enzyme-conjugation methods, this method allows the generation of far more amplified detection signals, thereby markedly increasing the sensitivity of detection enough to analyze a target present in aM concentrations.

A highly sensitive and versatile transcription immunoassay using a DNA-encoding tandem repetitive light-up aptamer.

Highly sensitive and accurate measurements of protein biomarkers are crucial for early diagnosis and disease monitoring. Here we report a versatile detection platform for sensitive detection of a protein biomarker using a tandem repeat Spinach aptamer DNA-based transcription immunoassay, which is a immunoassay combined with transcription-assisted Spinach RNA aptamer generation. We designed a DNA template encoding spa tandem repetitive Spinach sequence for enhanced generation of an RNA aptamer. The tandem repeated Spinach DNA template is consist of multiple monomeric units which is composed of T7 promoter, Spinach-2 and terminator. After in vitro transcription, the fluorescence signal from the 16R (nR, n = number of repeats) DNA template was enhanced up to ~ 15-fold compared to a single form (1R) DNA template. Using tandem repeat DNA, the proposed transcription immunoassay showed a limit of detection (LOD) of 37 aM, which is 103-fold lower than that of the conventional enzyme-linked immunosorbent assay (ELISA). The results demonstrate substantial promise for the ultrasensitive detection of various biological analytes using simple ELISA techniques. The high sensitivity and reliability of the proposed transcription immunoassay offer great promise for clinical assays.

Biomimetic Thermal-sensitive Multi-transform Actuator.

Controllable and miniaturised mechanical actuation is one of the main challenges facing various emerging technologies, such as soft robotics, drug delivery systems, and microfluidics. Here we introduce a simple method for constructing actuating devices with programmable complex motions. Thermally responsive hydrogels based on poly(N-isopropylacrylamide) (PNIPAM) and its functionalized derivatives (f-PNIPAM) were used to control the lower critical solution temperature (LCST) or the temperature at which the gel volume changes. Techniques for ultra-violet crosslinking the monomer solutions were developed to generate gel sheets with controllable crosslink density gradients that allowed bending actuation to specified curvatures by heating through the LCST. Simple molding processes were then used to construct multi-transform devices with complex shape changes, including a bioinspired artificial flower that shows blossoming and reverse blossoming with a change in temperature.

Transcription immunoassay: light-up RNA aptamer-based immunoassay using in vitro transcription.

Here, we present an ultra-enhanced immunoassay for sensitive and reliable biomarker detection using layer-by-layer assembly and transcription-assisted light-up aptamer generation to induce signal amplification. This dendrimer structure-based transcription immunoassay is ∼1500 times more sensitive than commercial fluorescence ELISA, achieving a detection limit of 108 aM.

Cascading Amplification of Immunoassay Signal by Cell-Free Expression of Firefly Luciferase from Detection Antibody-Conjugated DNA in an Escherichia coli Extract.

An expression immunoassay is a powerful technique that combines unique features of immunosorbent assays and cell-free protein synthesis. The main advantage of the expression immunoassay is a greatly amplified signal, whereas a conventional enzyme-linked immunosorbent assay (ELISA) employs a single enzyme molecule conjugated to a detection antibody to produce a measurable signal. Expression immunoassays utilize a DNA molecule conjugated to a target-bound antibody to generate multiple enzyme molecules that then produce the signal. To date, expression immunoassays have not been widely adopted due to the limited availability of efficient methods for translating antibody-conjugated DNA. We developed a highly efficient translation module for expression immunoassays using an Escherichia coli extract-based cell-free protein synthesis system. When we used our immunoassay technique to detect α-fetoprotein, we achieved a limit of detection of 7 fM. Given the outstanding sensitivity that can be obtained with only minimal modifications to the procedure of standard ELISA, we believe that this method will open up new possibilities for widespread application of expression immunoassays to ultrasensitive detection and diagnostics.

Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H2O2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

An optical fiber-based LSPR aptasensor for simple and rapid in-situ detection of ochratoxin A.

Label-free biosensing methods that rely on the use of localized surface plasmon resonance (LSPR) have attracted great attention as a result of their simplicity, high sensitivity, and relatively low cost. However, in-situ analysis of real samples using these techniques has remained challenging because colloidal nanoparticles (NPs) can be unstable at certain levels of pH and salt concentration. Even in the case of a chip-type LSPR sensor that can resolve the instability problem by employing NPs immobilized on the substrate, loading of a sample to sensor chip with exact volume control can be difficult for unskilled users. Herein, we report an optical-fiber-based LSPR aptasensor that can avoid these problems and serve as a portable and simple system for sensitive detection of a small mycotoxin, ochratoxin A (OTA), in real samples. The optical fiber coated with aptamer-modified gold nanorods (GNRs) is simply dipped into a solution containing OTA and subjected to LSPR analysis. Quantitative analysis of OTA is performed by measuring the spectral red shift of the LSPR peak of GNRs. Under optimized conditions, the LSPR peak shift displays a linear response (R2 = 0.9887) to OTA in the concentration range from 10pM to 100nM, with a limit of detection of 12.0pM (3S). The developed sensor shows a high selectivity for OTA over other mycotoxins such as zearalenone (ZEN) and ochratoxin B (OTB), and shows an accurate detection capability for OTA in real grape juice samples.

Single-Step LRET Aptasensor for Rapid Mycotoxin Detection.

Contamination of foods by mycotoxins is a common yet serious problem. Owing to the increase in consumption of fresh produce, consumers have become aware of food safety issues caused by mycotoxins. Therefore, rapid and sensitive mycotoxin detection is in great demand in fields such as food safety and public health. Here we report a single-step luminescence resonance energy transfer (LRET) aptasensor for mycotoxin detection. To accomplish the single-step sensor, our sensor was constructed by linking a quencher-labeled aptamer through a linker to the surface of upconversion nanoparticles (UCNPs). Our LRET aptasensor is composed of Mn2+-doped NaYF4:Yb3+,Er3+ UCNPs as the LRET donor, and black hole quencher 3 (BHQ3) as the acceptor. The maximum quenching efficiency is obtained by modulating the linker length, which controls the distance between the quencher and the UCNPs. Our distinctive design of LRET aptasensor allows detection of mycotoxins selectively in colored food samples within 10 min without multiple bioassay steps. We believe our single-step aptasensor has a significant potential for on-site detection of food contaminants, environmental pollutants, and biological metabolites.

Asymmetric Nanocrescent Antenna on Upconversion Nanocrystal.

Frequency upconversion activated with lanthanide has attracted attention in various real-world applications, because it is far simpler and more efficient than traditional nonlinear susceptibility-based frequency upconversion, such as second harmonic generation. However, the quantum yield of frequency upconversion of lanthanide-based upconversion nanocrystals remains inefficient for practical applications, and spatial control of upconverted emission is not yet developed. Here, we developed an asymmetric nanocrescent antenna on upconversion nanocrystal (ANAU) to deliver excitation light effectively to the core of upconversion nanocrystal by nanofocusing light and generating asymmetric frequency upconverted emission concentrated toward the tip region. ANAUs were fabricated by high-angle deposition (60°) of gold (Au) on the isolated upconversion nanoparticles supported by nanopillars then moved to refractive-index matched substrate for orientation-dependent upconversion luminescence analysis in the single-nanoparticle scale. We studied shape-dependent nanofocusing efficiency of nanocrescent antennae as a function of the tip-to-tip distance by modulating the deposition angle. The generation of asymmetric frequency upconverted emission toward the tip region was simulated by the asymmetric far-field radiation pattern of dipoles in the nanocrescent antenna and experimentally demonstrated by the orientation-dependent photon intensity of frequency upconverted emission of an ANAU. This finding provides a new way to improve frequency upconversion using an antenna, which locally increases the excitation light and generates the radiation power to certain directions for various applications.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A highly sensitive and widely adaptable plasmonic aptasensor using berberine for small-molecule detection.

Localized surface plasmon resonance (LSPR) biosensors allow label-free detection of small molecules in molecular binding events; however, they are limited by a relatively low sensitivity and narrow dynamic range. Here, we report highly sensitive small-molecule detection by LSPR peak shift exploiting the G-quadruplex (GQx) structure-binding characteristic of known GQx binders to enhance the LSPR signal of a plasmonic aptasensor. Six known GQx binders (thiazole orange, malachite green, crystal violet, zinc protoporphyrin IX, thioflavin T, and berberine) were tested for their ability to enhance the LSPR signal. Among these, berberine (BER) induced the largest LSPR peak shift by interacting with the GQx structure formed by the aptamer/target binding event on a gold nanorod surface. This specific binding performance was confirmed by the fluorescence signal of BER and through repeated cycles of BER addition and washing on the plasmonic sensing chip. The proposed plasmonic aptasensor respectively showed limit of detection (LOD) of 0.56, 0.63, 0.87 and 1.05 pM for ochratoxin A, aflatoxin B1, adenosine triphosphate and potassium ions, which was 1000-fold higher than that in BER-free condition, and a wide dynamic range from 10 pM to 10µM. In addition, the proposed LSPR aptasensor could effectively be used to quantitatively analyze small molecules in real samples.

A Localized Surface Plasmon Resonance (LSPR)-based, simple, receptor-free and regeneratable Hg(2+) detection system.

A simple, receptor-free and regeneratable Hg(2+) sensor, which utilizes localized surface plasmon resonance (LSPR) shifts of a gold nanorod (GNR), has been developed. Precipitation induced by coordination of Hg(2+) to citrate alters the local refractive index (RI) around the GNR surface on glass slide, promoting a red-shift in its LSPR absorption peak. This phenomenon is used to design a sensor that enables quantitative detection of Hg(2+) in the 1nM to 1mM concentration range with good linearity (0.9507 correlation coefficient) and limit of detection (LOD) is reached to 0.38nM. A high selectivity of this sensor for Hg(2+) is demonstrated by the specific LSPR red-shift of 27.67nm promoted by Hg(2+) in comparison to those caused by other metal ions. In addition, the reusability of the new sensor chip is shown by its successful reuse eight-times following successive washing/precipitation steps. Lastly, the sensor displays excellent recoveries in spiking test with real water samples, such as tap water, lake and river. The simple combination of precipitation of Hg(2+)-citrate complex and the LSPR red-shift has led to the design of a novel sensing strategy for Hg(2+) detection.

A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme.

An ultrasensitive, colorimetric and homogeneous strategy for aflatoxin B1 (AFB1) detection, which uses a DNA aptamer and two split DNAzyme halves, has been developed. Split halves of a hemin-binding DNAzymes is combined with an AFB1 aptamer to generate a homogeneous colorimetric sensor that undergoes an AFB1 induced DNA structural change. In the absence of AFB1, the split probes have peroxidase mimicking DNAzyme activity associated with catalysis of a color change reaction. Specific recognition of AFB1 by the aptamer component leads to structural deformation of the aptamer-DNAzyme complex, which causes splitting of the DNAzyme halves and a reduction in peroxidase mimicking activity. Therefore, a decrease of colorimetric signal arising from the catalytic process takes place upon in the presence of AFB1 in a concentration dependent manner in the 0.1-1.0 × 10(4) ng/mL range and with a colorimetric detection limit of 0.1 ng/mL. The new assay system exhibits high selectivity for AFB1 over other mycotoxins and can be employed detect the presence of AFB1 in ground corn samples. Overall, the strategy should serve as the basis for the development of rapid, simple and low-cost methods for detection of mycotoxins.

High-sensitivity detection of ATP using a localized surface plasmon resonance (LSPR) sensor and split aptamers.

A highly sensitive localized surface plasmon resonance (LSPR) aptasensor for detection of adenosine triphosphate (ATP) has been developed. The sensor utilizes two split ATP aptamers, one (receptor fragment) being covalently attached to the surface of a gold nanorod (GNR) and the other labeled with a random DNA sequence and TAMRA dye (probe fragment). In the presence of both ATP and the probe fragment, a significant shift takes place in the wavelength of the LSPR band. This phenomenon is a consequence of the fact that the split fragments assemble into an intact folded structure in the presence of ATP, which brings about a decrease in the distance between the GNR surface and TAMRA dye and an associated LSPR wavelength. By using this sensor system, concentrations of ATP in the range of 10 pM-10 µM can be determined. In addition, by taking advantage of its denaturation properties, the LSPR aptasensor can be reused by simply subjecting it to quadruple salt-addition/2M NaCl washing steps. That the new method is applicable to biological systems was demonstrated by its use to measure ATP concentrations in E. coli and, thus to determine cell concentrations as low as 1.0×10(3) CFU.