Isothermal Signal Amplification Research Articles

Sesame Detection in Food Using DNA-Functionalized Gold Nanoparticles: A Sensitive, Rapid, and Cost-Effective Colorimetric Approach.

Food safety control is a key issue in the food and agriculture industries. For such purposes, developing miniaturized analytical methods is critical for enabling the rapid and sensitive detection of food supplements, allergens, and pollutants. Here, a novel bioanalytical methodology based on DNA-functionalized gold nanoparticles (AuNPs) and colorimetric detection was developed to detect the presence of sesame (a major allergen) through sesame seed DNA as a target, in food samples. The presence of sesame DNA induces controlled nanoparticle aggregation/desegregation, resulting in a color change (from blue to red) proportional to sesame DNA concentration. The incorporation of multicomponent nucleic acid enzymes (MNAzymes) in this strategy has been carried out to perform an isothermal signal amplification strategy to improve the sensitivity of detection. Also, open-source software for color analysis was used to ensure an unbiased visual color-change detection, enhancing detection accuracy and sensitivity and opening the possibility of performing a simple and decentralized analyte detection. The method successfully detected the presence of sesame DNA in sesame seed, sesame oil, olive oil, and sunflower oil. In brief, the developed approach constitutes a simple and affordable alternative to perform a highly sensitive detection of DNA in food without complex methodologies or the requirement of expensive instrumentation.

High-Fidelity Sensitive Tracing Circulating Tumor Cell Telomerase Activity.

Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells·μL-1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics.

HcHCR: High-Throughput Single-Cell Imaging of RNA in Human Primary Immune Cells.

Functional genomics and chemical screens can identify and characterize novel cellular factors regulating signaling networks and chemical tools to modulate their function for the treatment of disease. Screening methods have relied primarily on immortalized and/or transformed cancer cell lines, which can limit the generalization of results to more physiologically relevant systems. Most have also relied on immunofluorescence, or on stably expressed recombinant fluorescent proteins, to detect specific protein markers using high-content imaging readouts. In comparison, high-throughput methods to visualize and measure RNA species have been less explored. To address this, we have adapted an isothermal signal amplification chemistry for RNA FISH known as hybridization chain reaction (HCR) to an automated, high-content imaging assay format. We present a detailed protocol for this technique, which we have named high-content HCR (hcHCR). The protocol focuses on the measurement of changes in mRNA abundance at the single-cell level in human primary cells, but it can be applied to a variety of primary cell types and perturbing agents. We anticipate that hcHCR will be most suitable for low- to medium-throughput screening experiments in which changes in transcript abundance are the desired output measure.

Ultrasound-enhanced catalytic hairpin assembly capable of ultrasensitive microRNA biosensing for the early screening of Alzheimer's disease

Catalytic hairpin assembly (CHA) is a promising enzyme-free, isothermal signal amplification strategy, but the relatively time-consuming strand replacement limits its application scenarios. Here, we developed an ultrasound-enhanced catalytic hairpin assembly (UECHA) biosensing platform for early screening of Alzheimer's disease by introducing a portable acoustic-drive platform with functionalized microspheres for effective biomarkers enrichment and fluorescence enhancement. By constructing a gradient ultrasonic field in a microcavity, the platform concentrates the functionalized microspheres in a central position, accompanied by an enhanced fluorescence signal with a specific release. In addition, the programmable frequency modulation can also modify the acoustic potential well and effectively promote non-equilibrium chemical reactions such as CHA (25 min). Compared with the conventional catalytic hairpin assembly (CHA), UECHA allows for direct and quantitative measurement of AD miRNAs down to 3.55 × 10−15 M in 1 μL samples. This visual analysis of ultra-trace biomarkers based on acoustic enrichment and promotion provides a new perspective for the rapid and highly sensitive clinical detection of Alzheimer's disease.

MNAzymes and gold nanoparticles as isothermal signal amplification strategy for visual detection of miRNA

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that are considered a novel emerging class of disease biomarkers in a variety of afflictions. Sensitive detection of miRNA is typically achieved using hybridization-based methods coupled with genetic amplification techniques. Although their sensitivity has improved, amplification techniques often present erroneous results due to their complexity. In addition, the use of these techniques is usually linked to the application of protein enzymes, the activity of which is dependent on the temperature and pH of the medium. To address these drawbacks, an alternative genetic enzyme for the highly sensitive detection of miRNAs is proposed in this work. Multicomponent nucleic acid enzymes (MNAzymes), coupled with the use of DNA-functionalized gold nanoparticles (AuNPs), were used in this study to develop an isothermal signal amplification strategy for visual genetic detection. miR146a, a biomarker of bovine mastitis present in milk, was selected as a model analyte. The developed methodology is easily carried out in 80 min at 50 °C, generating a low visual limit of detection of 250 pM based on the observation of a color change. The methodology was successfully applied to the detection of miR146a in raw cow milk samples.Graphical

DSN enzyme recognition initiated rolling circle amplification (DiRCA) for accurate miRNA detection in immune response and immune repair after trauma

Recent studies have reported that miRNA plays an important role in immune response and immune repair after trauma. By regulating the expression of related target genes, miRNA regulates the production, proliferation, development and immune response of immune cells. Therefore, it is in urgent demand to develop an novel method for miRNA analysis. Rolling circle amplification (RCA), as an attractive isothermal signal amplification strategy, has been widely utilized in constructing miRNA detection assays. However, accurate and sensitive miRNA quantitative determination remains a huge challenge for RCA based approaches. Herein, we propose a DSN enzyme based signal cycle initiated Rolling Circle Amplification assay (DiRCA) for sensitive and accurate miRNA detection. In DiRCA, target miRNA unfolds hairpin structure probe in the detection scaffold, forming a RNA–DNA duplex. DSN enzyme is utilized to specifically digest the DNA sequence in RNA–DNA duplex, releasing miRNA to form a signal cycle; its capability to distinguish one base pair mismatch in RNA–DNA duplex endows DiRCA a high selectivity. Meanwhile, DSN enzyme based cleavage initiates RCA, transcribing G-rich sequences for signal generation. Based on the DSN assisted signal cycle and RCA, DiRCA shows a low limit of detection of 0.43 fM and a superior capability in selectively detecting mismatched miRNA sequences, showing a promising prospect in the early-diagnosis of disease.

Retracted: Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection.

[This retracts the article DOI: 10.1155/2022/5989889.].

Intermolecular and Intramolecular Priming Co-directed Synergistic Multi-strand Displacement Amplification Empowers Ultrasensitive Determination of microRNAs.

The construction of isothermal nucleic acid amplification systems with extremely high signal amplification abilities is of great importance for biomarker detection and disease diagnosis. Herein, for the first time, we proposed an intermolecular and intramolecular priming co-directed synergistic multi-strand displacement amplification (SM-SDA) system for microRNA (miRNA) detection. Strategically, the SM-SDA system is made of a single multi-functionalized hairpin probe (MF-HP) that is engineered with a long stem and endowed with target complementation, configuration transformation, enzyme recognition, and signal reporting abilities. The presence of a specific target, the model of miRNA-21, to react with MF-HP would cause an intermolecular priming-directed strand replication in parallel with an intramolecular priming-directed strand replication. The co-directed priming pattern results in the occurrence of SM-SDA containing a target-induced cyclical strand displacement amplification (T-CSDA), a target analogue primer (TAP)-induced CSDA (TAP-CSDA), and a nicked trigger primer (NTP)-induced CSDA (NTP-CSDA). The resulting multiple circuits with a synergetic signal amplification capacity remarkably enhance the target miRNA response, which has not only improved the sensitivity for qualitative and quantitative detection of miRNA but also realized the analysis of target miRNA from real clinical samples. As a proof-of-concept study, this simple but attractive isothermal signal amplification system holds a great potential for molecular diagnosis of diseases and will stimulate interest, new ideas, and discoveries in this fascinating field.

CRISPR-Cas system manipulating nanoparticles signal transduction for cancer diagnosis.

Early diagnosis of cancer is important to improve the survival rate and relieve patient pain. Sensitive detection of cancer related biomarkers in body fluids is a critical approach for the early diagnosis of cancer. The clustered regularly interspaced short palindromic repeat-associated protein (CRISPR-Cas) system has emerged as a molecular manipulation technology because of its simple detection procedure, high base resolution, and isothermal signal amplification. Recently, various nanomaterials with unique optical and electrical characteristics have been introduced as the novel signal transducers to enhance the detection performance of CRISPR-Cas-based nanosensors. This review summarizes the working mechanisms of the CRISPR-Cas system for biosensing. It also enumerates the strategies of CRISPR-manipulated nanosensors based on various signal models for cancer diagnosis, including colorimetric, fluorescence, electrochemical, electrochemiluminescence, pressure, and other signals. Finally, the prospects and challenges of CRISPR-Cas-based nanosensors for cancer diagnostic are also discussed. This article is categorized under: Diagnostic Tools > Biosensing.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay

BackgroundIntegrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens.MethodsRT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored.ResultsThe results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/μL within approximately 30–40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement.ConclusionsTogether, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection.

Background: Isothermal signal amplification technique is developed based on the rolling ring amplification mechanism of cyclic DNA molecules in nature. This technique plays an extremely beneficial role in gonorrhea pathogen identification and drug resistance gene detection. Aims: This study analyzes the isothermal signal amplification techniques in the etiological diagnosis of gonorrhea and drug resistance gene detection. Materials and Methods: Urethral, cervical secretion, or prostatic fluid samples from 322 cases of gonorrhea collected from January 2018 to December 2021 at the STD clinic of our hospital dermatology department were selected for direct smear examination and gonococcal culture examination; DNA was extracted from urethral, cervical secretion, or prostatic fluid samples and then used for pathogen identification by SAT assay and rolling loop nucleic acid amplification technique, smear examination and pathogen culture examination methods, SAT assay, and isothermal signal amplification technique for comparative sensitivity and specificity analysis. Results: The highest rate of gonorrhea positivity was for the urine rolling loop nucleic acid amplification technique, followed by the swab rolling loop nucleic acid amplification technique, and the lowest rate of gonorrhea positivity was for the urine SAT test. The difference in the positivity rate between the two urine testing methods was statistically significant (P < 0.05). The highest sensitivity of the urine rolling loop nucleic acid amplification technique method for the detection of gonorrhea pathogens and the lowest sensitivity of the urine SAT method were statistically significant (P < 0.01). The differences in sensitivity and specificity between the swab rolling loop nucleic acid amplification technique and the swab SAT method were not statistically significant (P > 0.05). ROC curves were plotted based on sensitivity and specificity, with swab SAT assay (AUC = 0.998) > rolling loop nucleic acid amplification technique (AUC = 0.981). Comparing the negative rates of urine and swab rolling loop nucleic acid amplification technique and urine SAT assay, the differences were not statistically significant (P > 0.05). Conclusion: The isothermal signal amplification technique improves the shortcomings of gonorrhea pathogen identification means and drug resistance gene detection methods, with good detection sensitivity and specificity, simple operation, low price, and easy promotion, which has obvious advantages in clinical applications and epidemiological studies.

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

Purpose In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2.MethodsPrimers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed.ResultsThe limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively.ConclusionsThe results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11845-022-03046-2.

Hollow covalent organic framework-sheltering CRISPR/Cas12a as an in-vivo nanosensor for ATP imaging

In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.

Improving the robustness of a catalyzed hairpin assembly with a three-arm nanostructure for nonenzymatic signal amplification.

Kinetically trapped hairpin DNA has great potential to dynamically build nanostructures, which can be initiated by sequence-specific nucleic acids. The branched junction, which has a multi-arm structure, is a representative nanostructure of DNA. In this study, we report a nonenzymatic and isothermal signal amplification accompanied by building a 3-arm structure based on a catalyzed hairpin DNA assembly (3-CHA). We improved the signal-to-background ratio of the 3-CHA by suppressing the leakage pathway of 3-CHA, thus eliminating unfavorable reaction sites exposed in the single-stranded region of hairpin DNAs. Background and amplified signals were analyzed with gel electrophoresis and real-time fluorescence monitoring. The limit of detection of the developed 3-CHA was estimated to be 29.3 pM for catalyst DNA at room temperature. Supported by the reduced leakage signal, the implemented 3-CHA showed great potential for detecting low concentrations of target DNA.

Detection of Norovirus RNA based on catalytic hairpin assembly and magnetic separation of DNA AgNCs

Based on catalytic hairpin assembly (CHA) and magnetic separation, a fluorescence biosensor of Norovirus RNA was developed. In this detection strategy, these two isothermal signal amplification techniques as well as brightly fluorescent DNA-templated silver nanoclusters (AgNCs) of high quantum yield were employed, with the aim of highly sensitive detection of Norovirus RNA at room temperature. Results showed that the designed strategy of sensor could be successfully established and implemented. Under optimal reaction conditions, this sensor showed a good linear response towards the concentrations of target RNA in the range of 1.0–100 nM with a detection limit of 0.82 nM. A quenching phenomenon was found in this sensoring system, which was contributed together by the biotin-avidin complex and magnetic nanoparticles. The phenomenon was confirmed and explained for reference value of AgNCs based biosensors.

A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL−1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.

A universal CRISPR/Cas12a nucleic acid sensing platform based on proximity extension and transcription-unleashed self-supply crRNA

The extraordinary genome-editing tool CRISPR/Cas12a has also been utilized as a powerful sensing technology owing to its highly-specificity and isothermal signal amplification. Nevertheless, the widespread application of Cas12a-based sensing methods in nucleic acid detection is limited by the targeting range and high undesired background. Herein, we established a universal Cas12a-based nucleic acid sensing strategy by using proximity extension and transcription-unleashed self-suppling of crRNA. The target was recognized and bound to a pair of adjacent probes, and then triggered the proximity-induced primer extension and transcription amplification to produce numerous crRNAs. The amplified abundant crRNAs assembled with Cas12a and dsDNA activators containing PAM to form a ternary complex, which trans-cleaved ssDNA-FQ reporters continuously to generate a strong fluorescent signal. Thus, the cascade enzymatic amplification was performed and subsequently applied for detecting target DNA down to 41.7 amol with a low nonspecific background. The application of this strategy in RNA detection has also been demonstrated, and it is expected to provide a universal and sensitive sensing platform for molecular diagnosis applications.

The Recent Development of Hybridization Chain Reaction Strategies in Biosensors

With the continuous development of biosensors, researchers have focused increasing attention on various signal amplification strategies to pursue superior performance for more applications. In comparison with other signal amplification strategies, hybridization chain reaction (HCR) as a powerful signal amplification technique shows its certain charm owing to nonenzymatic and isothermal features. Recently, on the basis of conventional HCR, this technique has been developed and improved rapidly, and a variety of HCR-based biosensors with excellent performance have been reported. Herein, we present a systematic and critical review on the research progress of HCR in biosensors in the last five years, including the newly developed HCR strategies such as multibranched HCR, migration HCR, localized HCR, in situ HCR, netlike HCR, and so on, as well as the combination strategies of HCR with isothermal signal amplification techniques, nanomaterials, and functional DNA molecules. By illustrating some representative works, we also summarize the advantage and challenge of HCR in biosensors, and offer a deep discussion of the latest progress and future development trends of HCR in biosensors.

CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase

We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10−5 to 2.5 × 10−2 U/mL T4 PNKP) and a detection limit as low as 3.3 × 10−6 U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening.

A simple, sensitive and non-enzymatic signal amplification strategy driven by seesaw gate

Herein, a simple enzyme-free method based on the seesaw-gate-driven isothermal signal amplification strategy was developed for nucleic acid detection. In this method, a partially complementary double-stranded beacon was designed, after the addition of ssDNA or RNA of target sequence, the fluorescence signal was restored through a toehold-mediated strand displacement process, followed by a seesaw-like reaction with the aid of an auxiliary strand with the same length of the toehold domain. Liberation of the target would initiate the next round of seesaw reaction to achieve recycling amplification of the fluorescence signal. The method has the advantages of simple sequence design and free of any enzyme, which can realize rapid detection of the target at 25 °C with a detection limit of 9.8 pM for DNA and 83 pM for RNA. The potential applicability of the proposed method was also demonstrated, indicating that it can provide a fundamental strategy for the development of nucleic acid sensors.