Precise pathogen quantification by CRISPR-Cas: a sweet but tough nut to crack.

Pathogen detection is increasingly applied in medical diagnosis, food processing and safety, and environmental monitoring. Rapid, sensitive, and accurate pathogen quantification is the most critical prerequisite for assessing protocols and preventing risks. Among various methods evolved, those based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) have been developed as important pathogen detection strategies due to their distinct advantages of rapid target recognition, programmability, ultra-specificity, and potential for scalability of point-of-care testing (POCT). However, arguments and concerns on the quantitative capability of CRISPR-based strategies are ongoing. Herein, we systematically overview CRISPR-based pathogen quantification strategies according to the principles, properties, and application scenarios. Notably, we review future challenges and perspectives to address the of precise pathogen quantification by CRISPR-Cas. We hope the insights presented in this review will benefit development of CRISPR-based pathogen detection methods.

Hue-change coupled with CRISPR-Cas12a lateral flow assay for the semiquantitative detection of Salmo salar adulteration.

Salmo salar is one of the most popular salmon species due to its meaty texture and quality protein. Oncorhynchus mykiss, which has a muscle texture similar to that of Salmo salar and is less expensive, is often used as a substitute for Salmo salar. As Salmo salar and Oncorhynchus mykiss belong to the same subfamily of Salmonidae, traditional methods are ineffective in the specific detection of the two. In this study, we combined hue-change with CRISPR/Cas12a lateral flow assay to detect the Salmo salar adulteration. This method detected S. salar genomic DNA at a vLOD of 5 copies, and was able to accurately identify adulterated samples containing 5 % w/w Salmo salar within one hour. In addition, the detection of Salmo salar in processed food products was achieved with the naked-eye at a concentration range of 0 % âˆ¼ 70 % w/w, and the detection accuracy is between 93.3 % âˆ¼ 100 %.

Acoustofluidic precise manipulation: Recent advances in applications for micro/nano bioparticles.

Acoustofluidic technologies that integrate acoustic waves and microfluidic chips have been widely used in bioparticle manipulation. As a representative technology, acoustic tweezers have attracted significant attention due to their simple manufacturing, contact-free operation, and low energy consumption. Recently, acoustic tweezers have enabled the efficient and smart manipulation of biotargets with sizes covering millimeters (such as zebrafish) and nanometers (such as DNA). In addition to acoustic tweezers, other related acoustofluidic chips including acoustic separating, mixing, enriching, and transporting chips, have also emerged to be powerful platforms to manipulate micro/nano bioparticles (cells in blood, extracellular vesicles, liposomes, and so on). Accordingly, some interesting applications were also developed, such as smart sensing. In this review, we firstly introduce the principles of acoustic tweezers and various related technologies. Second, we compare and summarize recent applications of acoustofluidics in bioparticle manipulation and sensing. Finally, we outlook the future development direction from the perspectives such as device design and interdisciplinary.

Association between lower neutrophil-to-albumin ratio and early neurological improvement in patients with acute cerebral infarction after intravenous thrombolysis.

Elevated neutrophil counts and decreased albumin levels have been linked to an unfavorable prognosis in acute cerebral infarction (ACI). The objective of this study is to explore the correlation between the neutrophil-to-albumin ratio (NAR) and the early neurological improvement (ENI) of ACI patients following intravenous thrombolysis (IVT). ACI patients who underwent IVT between June 2019 and June 2023 were enrolled. The severity of ACI was assessed using the National Institutes of Health Stroke Scale (NIHSS). ENI was defined as a reduction in NIHSS score of ≥ 4 or complete resolution of neurological deficit within 24 h after IVT. Propensity score match (PSM) and logistic regression analysis were used to explore the correlation between these variables and the early neurological outcomes of patients. A total of 545 ACI patients were included, with 253 (46.4 %) experiencing ENI. Among the 193 pairs of patients after PSM, there was a significant association between NAR and ENI (OR, 0.89; 95 % CI, 0.85-0.94; p < 0.001). The restricted cubic splines analysis revealed a significant nonlinear correlation between NAR and ENI (p for nonlinear = 0.0004; p for overall = 0.0002). The optimal cutoff for predicting ENI was determined as a NAR level of 10.20, with sensitivity and specificity values of 73.6 % and 60.9 %. NAR levels are associated with ENI in ACI patients after IVT. The decreased levels of NAR indicate an increased likelihood of post-thrombolysis ENI in ACI patients.

Intensive Ambulance-Delivered Blood-Pressure Reduction in Hyperacute Stroke.

BACKGROUND: Treatment of acute stroke, before a distinction can be made between ischemic and hemorrhagic types, is challenging. Whether very early blood-pressure control in the ambulance improves outcomes among patients with undifferentiated acute stroke is uncertain. METHODS: We randomly assigned patients with suspected acute stroke that caused a motor deficit and with elevated systolic blood pressure (≥150 mm Hg), who were assessed in the ambulance within 2 hours after the onset of symptoms, to receive immediate treatment to lower the systolic blood pressure (target range, 130 to 140 mm Hg) (intervention group) or usual blood-pressure management (usual-care group). The primary efficacy outcome was functional status as assessed by the score on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) at 90 days after randomization. The primary safety outcome was any serious adverse event. RESULTS: A total of 2404 patients (mean age, 70 years) in China underwent randomization and provided consent for the trial: 1205 in the intervention group and 1199 in the usual-care group. The median time between symptom onset and randomization was 61 minutes (interquartile range, 41 to 93), and the mean blood pressure at randomization was 178/98 mm Hg. Stroke was subsequently confirmed by imaging in 2240 patients, of whom 1041 (46.5%) had a hemorrhagic stroke. At the time of patients' arrival at the hospital, the mean systolic blood pressure in the intervention group was 159 mm Hg, as compared with 170 mm Hg in the usual-care group. Overall, there was no difference in functional outcome between the two groups (common odds ratio, 1.00; 95% confidence interval [CI], 0.87 to 1.15), and the incidence of serious adverse events was similar in the two groups. Prehospital reduction of blood pressure was associated with a decrease in the odds of a poor functional outcome among patients with hemorrhagic stroke (common odds ratio, 0.75; 95% CI, 0.60 to 0.92) but an increase among patients with cerebral ischemia (common odds ratio, 1.30; 95% CI, 1.06 to 1.60). CONCLUSIONS: In this trial, prehospital blood-pressure reduction did not improve functional outcomes in a cohort of patients with undifferentiated acute stroke, of whom 46.5% subsequently received a diagnosis of hemorrhagic stroke. (Funded by the National Health and Medical Research Council of Australia and others; INTERACT4 ClinicalTrials.gov number, NCT03790800; Chinese Trial Registry number, ChiCTR1900020534.).

Microwave-Enabled Fast Preparation of a Metal-Organic Framework Hybrid Membrane for Filtration-Enhanced Simultaneous Separation and Detection of Aflatoxin B1.

Mycotoxin contamination in food and the environment seriously harms human health. Sensitive and timely detection of mycotoxins is crucial. Here, we report a dual-functional hybrid membrane with absorptivity and responsiveness for fluorescent-quantitative detection of mycotoxin aflatoxin B1 (AFB1). A biomineralization-inspired and microwave-accelerated fabrication method was established to prepare a hybrid membrane with a metal-organic framework (MOF) loaded in high density. The MOF presented high efficiency in capturing AFB1 and showed fluorescence intensity alteration simultaneously, enabling a dual adsorption-response mode. Deriving from the inherent porous structure of the hybrid membrane and the absorptive/responsive ability of the loaded MOF, a filtration-enhanced detection mode was elaborated to provide a 1.67-fold signal increase compared with the conventional soaking method. Therefore, the hybrid membrane exhibited a rapid response time of 10 min and a low detection limit of 0.757 ng mL-1, superior to most analogues in rapidity and sensitivity. The hybrid membrane also presented superior specificity, reproducibility, and anti-interference ability and even performed well in extreme environments such as strong acid or alkaline, satisfying the practical requirements for facile and in-field detection. Therefore, the membrane had strong applicability in chicken feed samples, with a detection recovery between 70.6% and 101%. The hybrid membrane should have significant prospects in the rapid and in-field inspection of mycotoxins for agriculture and food.

Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.

Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.

Association between fibrinogen-to-albumin ratio and functional prognosis of 3 months in patients with acute ischemic stroke after intravenous thrombolysis.

BACKGROUND: The presence of high fibrinogen and low albumin levels in serum is associated with a negative prognosis in acute ischemic stroke (AIS). Fibrinogen-to-albumin ratio (FAR), a new inflammatory biomarker, may provide better prognostic insights in patients with AIS than separate evaluation of fibrinogen or albumin. The objective of this investigation is to examine the correlation between FAR and 3-month functional prognosis after intravenous thrombolysis (IVT) in AIS patients. METHODS: The retrospective study recruited AIS patients who received IVT from June 2014 to December 2021. The 3-month functional prognosis was assessed using the Modified Rankin Scale (mRS). A mRS score of ≤2 indicated a good outcome, whereas a mRS score of >2 suggested a poor outcome. RESULTS: A total of 591 AIS patients who underwent IVT were included and 147 patients (24.9 %) had a poor outcome. Among the 102 pairs of patients after propensity score matching, there was a significant association between FAR and 3-month prognosis (adjusted OR, 1.19; 95% CI, 1.03-1.38; p = .020). The optimal FAR cutoff value was found to be 7.57, and even after stratifying patients based on this value, we still observed a significant correlation between high FAR level and poor outcome (adjusted OR, 2.08; 95% CI, 1.28-3.40; p = .003). CONCLUSIONS: FAR may serve as a prospective biomarker of predicting 3-month prognosis in AIS patients after IVT.

CRISPR-Cas12a-Mediated Hue-Recognition Lateral Flow Assay for Point-of-Need Detection of Salmonella.

Nucleic acid detection of pathogens in a point-of-need (PON) manner is of great significance yet remains challenging for sensitive and accurate visual discrimination. Here, we report a CRISPR-Cas12a-mediated lateral flow assay for PON detection of Salmonella typhimurium (S.ty) that is a prevailing pathogen disseminated through tainted food. The variation of the fluorescence color of the test line is exploited to interpret the results, enabling the discrimination between positive and negative samples on the basis of a hue-recognition mechanism. By leveraging the cleavage activity of Cas12a and hue-recognition readout, the assay facilitated by recombinase polymerase amplification can yield a visual detection limit of 1 copy µL-1 for S.ty genomic DNA within 1 h. The assay also displays a high specificity toward S.ty in fresh chicken samples, as well as a sensitivity 10-fold better than that of the commercial test strip. Moreover, a semiquantitative detection of S.ty ranging from 0 to 4 × 103 CFU/mL by the naked eye is made possible, thanks to the easily discernible color change of the test line. This approach provides an easy, rapid, accurate, and user-friendly solution for the PON detection of Salmonella and other pathogens.

Label-Free Sequence-Specific Visualization of LAMP Amplified Salmonella via DNA Machine Produces G-Quadruplex DNAzyme.

Salmonella is one of four key global causes of diarrhea, and in humans, it is generally contracted through the consumption of contaminated food. It is necessary to develop an accurate, simple, and rapid method to monitor Salmonella in the early phase. Herein, we developed a sequence-specific visualization method based on loop-mediated isothermal amplification (LAMP) for the detection of Salmonella in milk. With restriction endonuclease and nicking endonuclease, amplicons were produced into single-stranded triggers, which further promoted the generation of a G-quadruplex by a DNA machine. The G-quadruplex DNAzyme possesses peroxidase-like activity and catalyzes the color development of 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) as the readouts. The feasibility for real samples analysis was also confirmed with Salmonella spiked milk, and the sensitivity was 800 CFU/mL when observed with the naked eye. Using this method, the detection of Salmonella in milk can be completed within 1.5 h. Without the involvement of any sophisticated instrument, this specific colorimetric method can be a useful tool in resource-limited areas.

Enzymatic Catalysis in Size and Volume Dual-Confined Space of Integrated Nanochannel-Electrodes Chip for Enhanced Impedance Detection of Salmonella.

Nanochannel-based confinement effect is a fascinating signal transduction strategy for high-performance sensing, but only size confinement is focused on while other confinement effects are unexplored. Here, a highly integrated nanochannel-electrodes chip (INEC) is created and a size/volume-dual-confinement enzyme catalysis model for rapid and sensitive bacteria detection is developed. The INEC, by directly sandwiching a nanochannel chip (60 µm in thickness) in nanoporous gold layers, creates a micro-droplet-based confinement electrochemical cell (CEC). The size confinement of nanochannel promotes the urease catalysis efficiency to generate more ions, while the volume confinement of CEC significantly enriches ions by restricting diffusion. As a result, the INEC-based dual-confinement effects benefit a synergetic enhancement of the catalytic signal. A 11-times ion-strength-based impedance response is obtained within just 1 min when compared to the relevant open system. Combining this novel nanoconfinement effects with nanofiltration of INEC, a separation/signal amplification-integrated sensing strategy is further developed for Salmonella typhimurium detection. The biosensor realizes facile, rapid (<20 min), and specific signal readout with a detection limit of 9 CFU mL-1 in culturing solution, superior to most reports. This work may create a new paradigm for studying nanoconfined processes and contribute a new signal transduction technique for trace analysis application.

Recent advances in determination applications of emerging films based on nanomaterials.

Sensitive and facile detection of analytes is crucial in various fields such as agriculture production, food safety, clinical diagnosis and therapy, and environmental monitoring. However, the synergy of complicated sample pretreatment and detection is an urgent challenge. By integrating the inherent porosity, processability and flexibility of films and the diversified merits of nanomaterials, nanomaterial-based films have evolved as preferred candidates to meet the above challenge. Recent years have witnessed the flourishment of films-based detection technologies due to their unique porous structures and integrated physical/chemical merits, which favors the separation/collection and detection of analytes in a rapid, efficient and facile way. In particular, films based on nanomaterials consisting of 0D metal-organic framework particles, 1D nanofibers and carbon nanotubes, and 2D graphene and analogs have drawn increasing attention due to incorporating new properties from nanomaterials. This paper summarizes the progress of the fabrication of emerging films based on nanomaterials and their detection applications in recent five years, focusing on typical electrochemical and optical methods. Some new interesting applications, such as point-of-care testing, wearable devices and detection chips, are proposed and emphasized. This review will provide insights into the integration and processability of films based on nanomaterials, thus stimulate further contributions towards films based on nanomaterials for high-performance analytical-chemistry-related applications.

Acoustofluidic black holes for multifunctional in-droplet particle manipulation.

Acoustic black holes offer superior capabilities for slowing down and trapping acoustic waves for various applications such as metastructures, energy harvesting, and vibration and noise control. However, no studies have considered the linear and nonlinear effects of acoustic black holes on micro/nanoparticles in fluids. This study presents acoustofluidic black holes (AFBHs) that leverage controlled interactions between AFBH-trapped acoustic wave energy and particles in droplets to enable versatile particle manipulation functionalities, such as translation, concentration, and patterning of particles. We investigated the AFBH-enabled wave energy trapping and wavelength shrinking effects, as well as the trapped wave energy-induced acoustic radiation forces on particles and acoustic streaming in droplets. This study not only fills the gap between the emerging fields of acoustofluidics and acoustic black holes but also leads to a class of AFBH-based in-droplet particle manipulation toolsets with great potential for many applications, such as biosensing, point-of-care testing, and drug screening.

Improving the Prehospital Identification and Acute Care of Acute Stroke Patients: A Quality Improvement Project.

BACKGROUND: There are a large number of stroke patients in China, and there is currently a lack of prehospital acute stroke care training programs. AIM: To develop a prehospital emergency medical service (PEMS) training program to improve the prehospital identification and acute care of acute stroke. METHODS: Forty prehospital emergency doctors whose service stations are located within a 10 km radius from Shanghai Pudong New Area Medical Emergency Service Center took this course on November 13, 2014. A questionnaire was designed to evaluate the PEMS personnel's knowledge in stroke and acute stroke care and was conducted before and after training as an assessment of the effectiveness of training. The patient population in this study included a baseline cohort before training and a prospective cohort after training, each composed of patients who were sent to Shanghai East Hospital South Stoke Center within one year. The transit time, final diagnosis, administration of thrombolysis, and door-to-needle time (DNT) were collected and analyzed. RESULTS: After the training, 100% of the PEMS personnel were competent to identify stroke cases using the Cincinnati prehospital stroke scale (CPSS). All participants realized that intravenous thrombolysis therapy in a time-sensitive manner is the most effective way to treat acute ischemic stroke. Although there was no difference in first-aid transit time before and after training, the stroke diagnosis rate improved by 6.5% after training (P=0.03). The thrombolysis rate increased to 29.6% from 24.3% but did not reach statistical significance. Compared to 84.0 minutes (standard deviation: 23.1 minutes) before the training, the average DNT after training was 53 minutes (standard deviation: 15.0 minutes), demonstrating a remarkable reduction (P < 0.01). CONCLUSION: The training program effectively improved the PEMS personnel's knowledge in stroke and stroke acute care.

Rapid, Visual, and Sequence-Specific Detection of Salmonella in Egg Liquid with vis-NEAA, a CRISPR/Cas12 Empowered New Strategy.

Salmonella is one of the main pathogenic factors that cause foodborne diseases. Rapid and accurate detection of Salmonella in food is of great importance to ensure food safety. Nicking enzyme-assisted amplification (NEAA) is one of the promising isothermal amplification methods finishing the in vitro amplification in ∼10 min; however, it suffers from nonspecific amplification a lot (∼70% products are noises). In this paper, we introduced CRISPR/Cas12a to specifically recognize the NEAA amplicons and transduce the signals into turned-on fluorescent visual readouts (vis-NEAA). Impressively, with this method, the high efficiency of NEAA has been taken great advantage and the nonspecific products were successfully bypassed at the same time. In comparison to NEAA-gel electrophoresis, vis-NEAA showed complete fidelity toward the presence of specific products, while for real-time PCR, it possesses equivalent sensitivity and specificity but saves ∼80% of the time. A level of 80 CFU/mL Salmonella in spiked eggs can be detected on-site in ∼20 min.

Engineering DNA G-quadruplex assembly for label-free detection of Ochratoxin A in colorimetric and fluorescent dual modes.

Colorimetric and fluorescent methods for Ochratoxin A (OTA) detection are convenient and well received. However, the pigments and autofluorescence originated from food matrices often interfere with detection signals. We have developed a strategy with colorimetric and fluorescent dual modes to solve this challenge. In the colorimetric mode, OTA aptamer (AP9) was assembled into a DNA triple-helix switch with a specially designed signal-amplifying sequence. The OTA-induced G-quadruplex (G4) of AP9 would open the switch and release the signal-amplifying sequence for colorimetric signal amplification. The G4 structures of AP9 were further utilized to combine with the fluorogenic dye ThT for fluorescent mode. By skillfully engineering DNA G4 assembly for signal amplification, there was no need for any DNA amplification or nanomaterials labeling. Detections could be carried out in a wide temperature range (22-37 â„ƒ) and finished rapidly (colorimetric mode, 60 min; fluorescent mode, 15 min). Broad linear ranges (colorimetric mode, 10-1.5 ×103 µg/kg; fluorescent mode, 0.05-1.0 ×103 µg/kg) were achieved. The limit of detection for colorimetric and fluorescent modes were 4 µg/kg and 0.01 µg/kg, respectively. The two modes have been successfully applied to detect OTA in samples with intrinsic pigments and autofluorescence, showing their applicability and reliability.

Recent progress in visual methods for aflatoxin detection.

Aflatoxins (AFs) contamination in food and agricultural products poses a significant threat to human health. Sensitive and accurate detection of AFs provides a strong guarantee for ensuring food safety. Conventional chromatographic-based or mass spectrum methods, which rely on bulky instrument and skilled personnel, are not suitable for on-site surveillance. By contrast, visual detections which possess the merits of rapidity and sophisticated instrument-free present an excellent potential for the on-site detection of AFs. This review intends to summarize the latest development of visual methods for AFs detection, including paper-based tests, chromogenic reactions, and luminescent methods. Emerging technologies, like nanotechnology, DNAzymes, and aptamers combined with these visual methods are introduced. The basic principles, features, and application advantages of each type of visual methods are discussed. The biggest challenges and perspectives on their future trends are also addressed.

Mechanical Thrombectomy for Acute Stroke Due to Large-Vessel Occlusion Presenting With Mild Symptoms.

Purpose: To evaluate the safety and efficacy of mechanical thrombectomy (MT) for acute stroke due to large vessel occlusion (LVO), presenting with mild symptoms. Methods: A prospective cohort study of patients with mild ischemic stroke and LVO was conducted. Patients were divided into two groups: MT group or best medical management (MM) group. Propensity score matching (PSM) was conducted to reduce the confounding bias between the groups. The primary outcome was functional independence at 90 days. The safety outcome was symptomatic intracranial hemorrhage (sICH). Univariate and multivariate logistic regression analyses were used to identify the independent factors associated with outcomes. Results: Among the 105 included patients, 43 were in the MT group and 62 in the MM group. Forty-three pairs of patients were generated after PSM. There were no significant differences in sICH rates between two groups (p = 1.000). The MT group had a higher proportion of independent outcomes (83.7% MT vs. 67.4% MM; OR 2.483; 95% CI 0.886-6.959; p = 0.079) and excellent outcomes (76.7% MT vs. 51.2% MM; OR 3.150; 95% CI 1.247-7.954; p = 0.013) compared to the MM group, especially in patients with stroke of the anterior circulation (p < 0.05). Multivariate logistic regression analysis showed that small infarct core volume (p = 0.015) and MT treatment (p = 0.013) were independently associated with excellent outcomes. Conclusions: Our results suggest that MT in stroke patients, presenting with mild symptoms, due to acute LVO in the anterior circulation may be associated with satisfactory clinical outcomes. Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT04526756.

Label-Free Colorimetric Method for Detection of Vibrio parahaemolyticus by Trimming the G-Quadruplex DNAzyme with CRISPR/Cas12a.

Vibrio parahaemolyticus (V. parahaemolyticus), which may cause gastrointestinal disorders in humans, is a pathogen commonly found in seafood. There are many methods for detecting V. parahaemolyticus, yet they have some shortcomings, such as high cost, labor-intensiveness, and complicated operation, which are impractical for resource-limited settings. Herein, we present a sequence-specific, label-free, and colorimetric method for visual detection of V. parahaemolyticus. This method utilizes CRISPR/Cas12a to specifically recognize the loop-mediated isothermal amplification (LAMP) products for further trans-cleaving the G-quadruplex DNAzyme and depriving its peroxidase-mimicking activity. In this way, the results can be directly observed with the naked eyes via the color development of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS2-), which displays colorless for positive samples while green for target-free samples. We term such Cas12a-crRNA preventing ABTS2- from developing color by trimming the G-quadruplex DNAzyme as Cascade. The proposed method can detect 9.8 CFU (per reaction) of pure cultured V. parahaemolyticus, and the sensitivity is comparable to real-time LAMP. It has been applied for practical use and showed the capability to detect 6.1 × 102 CFU/mL V. parahaemolyticus in shrimp samples. Based on this, the newly established Cascade method can be employed as a universal biosensing strategy for pathogenic bacterial testing in the field.

Visual detection of in vitro nucleic acid replication by submicro- and nano-sized materials.

The rapid growth of in vitro nucleic acid replication has offered a powerful tool for clinical diagnosis, food safety detection and environmental monitorning. Successful implementation of various isothermal nucleic acid amplification methods enables rapid replication of target sequences without the participant of a thermal cycler. Point-of-need analysis possesses great superiorities in user-friendly, instant results analysis, low manufacturing, and consumable costs. To meet the great challenge of point-of-need analysis, developing simple and rapid visual methods becomes crucial. Submicro- and nanomaterials possess unique surface properties, which enables their rapid response to DNA amplicons. Their unique optical, magnetic, catalytic, and other physical/chemical properties have been frequently employed for the visual detection of in vitro nucleic acid replications. Herein, we aim to review the submicro- and nanomaterials-based visual methods for detection of nucleic acid amplification. The visual methods are classified according to the designing strategies (e.g. LSPR, bridging flocculation, luminescence, catalytic reaction, separation, etc.). The basic principles, merits and drawbacks of each strategy are described. The application in analysis of nucleic acid targets and non-nucleic acid targets are discussed. The main challenges and future research directions are also highlighted in this rapidly emerging field.