Real-time Fluorescence Detection Research Articles

Face-to-face Assembly Strategy of Au Nanocubes: Induced Generation of Broad Hotspot Regions for SERS-Fluorescence Dual-Signal Detection of Intracellular miRNAs.

While designing anisotropic noble metal nanoparticles (NPs) can enhance the signal intensity of Raman dyes, more sensitive surface-enhanced Raman scattering (SERS) probes can be designed by oriented self-assembly of noble metal nanomaterials into dimers or higher-order nanoclusters. In this study, we engineered a self-assembly strategy in living cells for real-time fluorescence and SERS dual-channel detection of intracellular microRNAs (miRNAs), using Mg2+-dependent 8-17E DNAzyme sequences as the driving motors, gold nanocubes (AuNCs) as the driver components, and three-branched double-stranded DNA as the linking tool. The assembly selects adenine in DNA as a reporter molecule, simplifying the labeling process of Raman reporter molecules and reducing the synthesis process. In addition, adenine is stably distributed between the faces of AuNCs and the wide hotspot region gives good reproducibility of the adenine SERS signal. In this strategy, the SERS channel was consistently stable and more sensitive compared to the fluorescence channel. Among them, the detection limit of the SERS channel was 2.1 pM and the coefficient of variation was 1.26% in the in vitro liquid phase and 1.49% in MCF-7 cells. The strategy successfully achieved accurate tracking and quantification of miRNA-21 in cancer cells, showing good reproducibility in complex samples as well as cells. The reported strategy provides ideas for exploring intracellular specific triggering of nanoparticles for precise control of self-assembly.

Brilliant quantum dots’ photoluminescence from a dual-resonance plasmonic grating

Semiconductor quantum dots (QDs) have recently caused a stir as a promising and powerful lighting material applied in real-time fluorescence detection, display, and imaging. Photonic nanostructures are well suited for enhancing photoluminescence (PL) due to their ability to tailor the electromagnetic field, which raises both radiative and nonradiative decay rate of QDs nearby. However, several proposed structures with a complicated manufacturing process or low PL enhancement hinder their application and commercialization. Here, we present two kinds of dual-resonance gratings to effectively improve PL enhancement and propose a facile fabrication method based on holographic lithography. A maximum of 220-fold PL enhancement from CdSe/CdS/ZnS QDs are realized on 1D Al-coated photoresist (PR) gratings, where dual resonance bands are excited to simultaneously overlap the absorption and emission bands of QDs, much larger than those of some reported structures. Giant PL enhancement realized by cost-effective method further suggests the potential of better developing the nanostructure to QD-based optical and optoelectronic devices.

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3-4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

An integrated low-cost automatic pipetting used in nucleic acid detection PCR instrument and clinical detection

Purpose This paper aims to introduce a custom-designed integrated nucleic acid detection polymerase chain reaction (PCR) instrument for clinical detection applications. Design/methodology/approach The PCR instrument can make rapid, sensitive, low-cost and quantitative molecular diagnosis compared with the current routine test flow from the pipette, series reagent to RT-PCR by manual manipulation. By integrating the multichannel automatic pipetting module, heat amplification module and real-time fluorescence detection module for the first time, the custom-designed integrated nucleic acid detection PCR instrument can achieve sample collection, subpackage, mixing, extracting, measuring and result presentation. Findings The multichannel automatic pipetting module was assembled with an accuracy of 0.4% (2 microliters) for accuracy measurement. Besides, the accuracy and sensitivity of nucleic acid using integrated low-cost nucleic acid detection PCR instruments were checked with COV-2019 virus (staining method) and African swine fever virus (probe method) under different concentrations. Practical implications Because of its high cost, complex system and bulky laboratory settings, including sample subpackage, mixing, extracting, measuring and finally result in presentation, the current nucleic acid detection system is not suitable for field operation and disease diagnosis in remote areas. The group independently designed and assembled an integrated low-cost multichannel nucleic acid detection PCR instrument, including a multichannel automatic pipetting module, a heat amplification module and a real-time fluorescence detection module. Originality/value The above equipment showed better reliability compared with commercial qPCR. These results can lay the foundation for functional, fast and low-cost PCR equipment for trace measurements.

Acceptability of self-collected vaginal samples for human papillomavirus testing for primary cervical cancer screening: comparison of face-to-face and online recruitment modes

BackgroundThis study aimed to assess the acceptability and attitudes of women towards human papillomavirus (HPV) self-sampling and compare the effectiveness of two delivery modes utilising face-to-face and online website for cervical cancer screening in Hong Kong.MethodsWomen aged 30–65 years were invited to participate by distributing the study information pamphlets at the specialist clinics of a regional acute hospital. Those who were interested in participating were given the option to join directly face-to-face or through an online website. All participants provided informed consent and received self-sampling kits and acceptability questionnaires either immediately (face-to-face) or through the post after registering at the website (online). All participants were requested to collect their own vaginal samples using a swab which was then brushed on a DNA sample storage card and returned to the hospital either in person or by post. The self-collected samples were tested for high-risk HPV using the Sentis™ HPV assay, a validated isothermal nucleic acid amplification real-time fluorescent detection assay. The primary outcome was the uptake rate of HPV self-sampling.ResultsOf the 1998 women recruited (1200 face-to-face, 798 online), 1377 returned their samples, giving an overall uptake rate of 68.9%. The uptake rate was significantly greater in the face-to-face mode than in the online mode (74.6% vs. 60.4%, p < 0.001). The median age of the participants was 49 years, 43.7% were never or under-screened, and 7.1% had high-risk HPV detected. Overall, 82.1% of the participants reported self-sampling convenient, and 79.3% were not embarrassed when collecting self-samples. However, only 49.8% were confident that they had collected the self-samples correctly. Most (91.1%) of the participants expressed willingness to perform self-sampling again, mostly because it was simple (79.2%) and quick (56.3%).ConclusionsHPV self-sampling can serve as an alternative primary screening method for cervical cancer in Hong Kong, especially for individuals who have not been adequately screened in the past. Both face-to-face and online website recruitment were associated with high acceptability, emphasising the potential benefits of utilising different platforms and strategies for reaching diverse populations.

A novel point-of-care platform for rapid SARS-CoV-2 detection utilizing an all-in-one 3D-printed microfluidic cartridge and IoT technology

The ongoing COVID-19 pandemic has created a significant demand for diagnostic tests, leading to shortages in medical supplies and hindering efforts to control infection outbreaks. We developed a novel point-of-care (POC) molecular diagnostic platform for SARS-CoV-2 identification utilizing a self-contained 3D-printed microfluidic cartridge and an internet-of-things-based diagnostic device. The platform enables automated downstream processes including magnetic bead-based RNA extraction, precise liquid aliquoting into four micro-reactors, accurate temperature control for reverse transcription-loop mediated isothermal amplification reaction, real-time fluorescence detection by a CMOS sensor, and data processing and display. The whole process is completed within 70 minutes with a limit of detection of 1×10−1 copies/μl in a miniaturized size (16 cm [height] × 12 cm [width] × 9 cm [length]). This advanced POC platform is simple and low-cost for manufacturing with 3D-printing technology and can play a crucial role in addressing medical supply shortages and relieving the urgent demand for COVID-19 tests.

Establishment of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus.

Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/μl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV.

Multi-DNA-Modified Double-Network Hydrogel with Customized Microstructure: A Novel System for Living Circulating Tumor Cells Capture and Real-Time Detection.

The precise and effective isolation of living circulating tumor cells (CTCs) from peripheral blood, followed by their real-time monitoring, is crucial for diagnosing cancer patients. In this study, a cell-imprinted double-network (DN) hydrogel modified with circular multi-DNA (CMD), coined the CMD-imprinted hydrogel with fixed cells as templates (CMD-CIDH), was developed. The hydrogel featured a customized surface for proficient capture of viable CTCs and in situ real-time fluorescent detection without subsequent release. The customized surface, constructed using polyacrylamide/chitosan DN hydrogel as the matrix on the cell template, had a dense network structure, thereby ensuring excellent stability and a low degradation rate. Optimal capture efficiencies, recorded at 93 ± 3% for MCF-7 cells and 90 ± 2% for Hela cells, were achieved by grafting the CMD and adjusting the nodule size on the customized surface. The capture efficiency remained significantly high at 67 ± 11% in simulated breast cancer patient experiments even at a minimal concentration of 5 cells mL-1. Furthermore, CMD grafted onto the surface produced a potent fluorescence signature, enabling in situ real-time fluorescent detection of the target cell's growth state even in complex environments. The customized surface is highly efficient for screening CTCs in peripheral blood and has promising potential for setting up the CTCs culture.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens

Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing.

The ongoing multi-country outbreak of monkeypox virus (MPXV) has continuously attracted global attention, highlighting the critical need for timely and accurate methods to detect MPXV and differentiate its clades. Herein, we devised a novel multiplex ET-PCR (endonuclease restriction-mediated real-time PCR) assay that integrates PCR amplification, restriction endonuclease cleavage and real-time fluorescence detection to diagnose MPXV infection and distinguish the Congo Basin and West African MPXV strains. In the MPXV ET-PCR system, three sets of specific primers were designed for MPXV, Congo Basin and West African strains. A short sequence, which could be recognized by restriction endonuclease enzyme BstUI, was added to the 5'end of amplification primers. Then, the modified primers were assigned different reporter dyes and corresponding quenching dyes to each of the three targets, enabling real-time fluorescence reporting of the results and multiplex detection. The designed assay enabled the detection of single or three targets in a single tube, with excellent specificity and analytical sensitivity in terms of plasmid and pseudotyped virus. Moreover, the clinical feasibility of our assay was validated using artificially simulated plasma, nasopharyngeal swab and skin swab samples. In conclusion, the multiplex ET-PCR assay devised here had the advantages of simple primer design, cost-effectiveness, low contamination risk, excellent sensitivity, high specificity and multiplex detection, making it a valuable and dependable tool for curbing the extensive spread of MPXV.

Steady Decline of HBV DNA Load under NAs in Lymphoma Patients and a Higher Level of qAnti-HBc Predict HBV Reactivation.

Background: Patients with lymphoma and chronic hepatitis B virus infection need to be treated with both chemotherapy and nucleotide analogue (NA) therapy. However, dynamic changes in HBV DNA loads with increasing chemotherapy cycles are lacking. It is unknown whether HBV replication markers, namely, the quantitative hepatitis B core antibody (qAnti-HBc), HBV RNA, and the hepatitis B virus core-related antigen (HBcrAg), are also markers for predicting HBV reactivation (HBVr). Methods: From 29 June 2010 to 6 December 2021, the data of patients with single-site diffuse large B-cell lymphoma and HBV infection (HBsAg+ and HBsAg-/anti-HBc+) were collected from a hospital medical record system, retrospectively. Serum HBV DNA loads (using real-time fluorescent quantitative PCR tests), qAnti-HBc levels (using a newly developed chemiluminescent particle immunoassay), HBV RNA levels (using the simultaneous amplification testing method based on real-time fluorescence detection), and HBcrAg levels (using a Lumipulse G HBcrAg assay) were tested, and factors related to HBVr were analyzed. Results: Under NAs, the HBV DNA loads of 69 HBsAg+ lymphoma patients declined from 3.15 (2.13-4.73) lg IU/mL to 1.00 (1.00-1.75) lg IU/mL, and further declined to 1.00 (1.00-1.04) lg IU/mL at the end of a 24-month follow-up. The qAnti-HBc levels decreased gradually during chemotherapy in HBsAg+ lymphoma patients (F = 7.090, p = 0.009). The HBV RNA and HBcrAg levels remained stable. A multivariate analysis revealed that higher qAnti-HBc levels (1.97 ± 1.20 vs. 1.12 ± 0.84 lg IU/mL, OR = 6.369, [95% CI: 1.523-26.641], p = 0.011) and higher HBV RNA levels (1.00 ± 1.13 vs. 0.37 ± 0.80 lg copies/mL, OR = 3.299, [95% CI: 1.229-8.854], p = 0.018) were related to HBVr in HBsAg-/anti-HBc+ lymphoma patients. Conclusions: HBV DNA loads declined under NAs during chemotherapy in lymphoma patients. In HBsAg-/anti-HBc+ lymphoma patients, a higher level of baseline serum qAnti-HBc and HBV RNA levels can predict the likelihood of HBVr during chemotherapy.

A simple AIE probe to pesticide trifluralin residues in aqueous phase: Ultra-fast response, high sensitivity, and quantitative detection utilizing a portable platform

The threat from pesticide trifluralin residues to ecological environment and public health is becoming a growing problem. Thus, rapid and sensitive detection, particularly a simple and portable detected platform for trifluralin residues, are highly desired. Here, a small organic aggregation-induced emission (AIE) molecule (TPETPy) is facilely synthesized and applied to detect trifluralin both in lab and in actual water systems. Based on the photo-induced electron transfer (PET) mechanism, the emissive peak of TPETPy located at 475 nm in tetrahydrofuran (THF)/water mixture (ƒw = 90 %) under the excitation of 340 nm, decreases dramatically upon trace trifluralin addition and exhibits ultra-fast response (3 s), high sensitivity and selectivity, and good anti-interference ability. The fluorescence sensing correlation with the concentration of trifluralin shows good linearity in the range of 20–90 μg L−1 with the limit of detection of 6.28 μg L−1. Moreover, a portable smartphone-integrated detected platform based on fluorescent pattern Red/Green/Blue (RGB) values is first employed to realize the real-time and on-site quantitative fluorescent detection of trifluralin in actual water sources, featuring good accuracy and reproducibility. Hereby, this work provides not only a highly efficient trifluralin residues fluorescent probe but also a portable and straightforward operating platform to detect trifluralin pesticides quantitatively.

An ultrafast and portable nucleic acid detection system based on photothermal PCR and real-time fluorescence detection

The nucleic acid detection technology based on polymerase chain reaction (PCR) is widely used in infectious disease detection due to its excellent sensitivity and specificity. However, the drawbacks of this method include bulky instrumentation in centralized laboratories and an assay time of 1–2 h. Here, we show a portable nucleic acid detection system, successfully performing ultrafast thermocycling and real-time fluorescence detection in a glass microtubule. By using plasmonic nanoparticles, the system can achieve 30 thermocycles in ∼ 2.5 min. The nucleic acid detection efficiency of the system is comparable to that of the conventional thermocycler. The system correctly distinguished the positive and negative samples of the clinical hepatitis B virus (HBV). Overall, this study extends PCR technology to point-of-care use and provides ideas for developing new virus detection systems.

High-throughput centrifugal microfluidic platform for multiplex respiratory virus diagnostics

We developed an advanced point-of-care (POC) molecular diagnostic platform that utilizes a centrifugal microfluidic chip for automating the diagnostics process of prevalent respiratory viruses, including Influenza A (H1N1, H3N2), Influenza B, and SARS-CoV-2, in a high-throughput (HTP) manner. The proposed POC microsystem can perform not only the whole processes including RNA purification, the reverse transcription loop-mediated isothermal amplification (RT-LAMP), and the real-time fluorescence detection, but also 30 sample analysis simultaneously in one run. Owing to the innovative zigzag aliquoting structure on the centrifugal microfluidic chip, the ultra-rapid and equivalent reagent division into 30 chambers with a single shot injection were possible, which expedited the high-throughput RNA extraction. The purified RNA templates were mixed with lyophilized RT-LAMP reagents and directed to the reaction chambers, where the primer sets for each target respiratory virus were freeze-dried for isothermal amplification. A total of 150 reactions (5 reaction chambers/unit × 30 units) were carried out on a single chip, so multiple and high-throughput diagnostics for 30 clinical samples could be completed in 1.5 hrs. Our platform with the high-throughput screening capability could be operated automatically, making it an invaluable tool for swift responses to viral pandemics such as COVID-19.

An Integrated Microfluidic Biosensing System Based on a Versatile Valve and Recombinase Polymerase Amplification for Rapid and Sensitive Detection of Salmonella typhimurium.

Detecting foodborne pathogens on-site is crucial for ensuring food safety, necessitating the development of rapid, cost-effective, highly sensitive, and portable devices. This paper presents an integrated microfluidic biosensing system designed for the rapid and sensitive detection of Salmonella typhimurium (S. typhimurium). The biosensing system comprises a microfluidic chip with a versatile valve, a recombinase polymerase amplification (RPA) for nucleic acid detection, and a customized real-time fluorescence detection system. The versatile valve combines the functions of an active valve and a magnetic actuation mixer, enabling on-demand mixing and controlling fluid flow. Quantitative fluorescence is processed and detected through a custom-built smartphone application. The proposed integrated microfluidic biosensing system could detect Salmonella at concentrations as low as 1.0 × 102 copies/µL within 30 min, which was consistent with the results obtained from the real-time quantitative polymerase chain reaction (qPCR) tests. With its versatile valve, this integrated microfluidic biosensing system holds significant potential for on-site detection of foodborne pathogens.

Combination of a robotic solution pipetting device with a centrifugal molecular diagnostic platform for fully automatic respiratory infectious virus detection from nasopharyngeal swab samples

A novel molecular diagnostic platform is presented that enables complete automation for the genetic analysis of respiratory infectious virus including the solution loading, RNA purification, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and real-time fluorescence detection. The overall structure consists of three parts: an integrated centrifugal microfluidic disc, a point-of-care (POC) genetic analyzer, and a robotic solution pipetting device. The integrated centrifugal microfluidic disc was designed to treat up to four samples simultaneously, with the capacity to detect seven targets in a single run. Additionally, a solution-loading cartridge is combined on the disc, facilitating the insertion of a nasopharyngeal swab sample as well as the storage and aliquoting of the washing solution, the elution solution, and the RT-LAMP master mix. The POC genetic analyzer is composed of a spinning motor for the automatic release of solutions, a couple of peltier heaters for gene amplification, a miniaturized fluorescence detector to generate an amplification curve, and an LED screen for displaying the experimental parameters and the diagnostic results. Importantly, we also integrate a robotic solution pipetting device with the POC genetic analyzer so that the designated solution can be loaded into the solution-loading cartridge without need of manual operation. Five targets (influenza A subtypes H1N1 and H3N2, influenza B, and SARS-CoV-2 (orf1ab and S gene)) of four samples were identified in 1.5 hr on a single device with a sample-in-answer-out manner.

Development of Real-Time Fluorescence CRISPR/Cas12a-Based Detection as a Portable Diagnostic System Using Integrated Circuits

A solution for achieving high-performance measurements in a space-constrained experimental setup was developed as a portable incubating instrument for real-time fluorescence detection of AvrPi9 gene in rice blast fungus by using a calibrated spectrometer in CRISPR-Cas12a detection. The system demonstrates accurate temperature control with low energy consumption and low deviation of ±0.16 °C from the setpoint temperatures, with high sensitivity and accurate detection within 10 min. The CRISPR-Cas12a detection reaction was demonstrated using AvrPi9 PCR product, crRNAs, LbCas12a and fluorescence-quencher reporter incubating at 37 °C for 10 min. Calibrated C12666MA spectrometer with 480 nm and 520 nm LEDs vs HR4000 reference exhibits low RMS of 0.54 and 1.30 and drift of 6.4 nm and 4.84 nm, respectively indicating high accuracy and reliability in fluorescence detection. Fluorescence signals were observed under an LED transilluminator, while real-time analysis was conducted through spectrometric measurements upon excitation by a 480 nm high-intensity blue LED source. Accuracy of detection between positive, non-template and non-target control was reported with no incidence of false positives observed. The instrument exhibits reliable quantitative detection capabilities with a limit of detection of 3.8 ng of DNA targets that are comparable to when running the same reaction on a commercial real-time PCR, with a detection limit of 1 ng. This study demonstrates that the CRISPR-Cas12a detection method represents a significant breakthrough in molecular diagnostics due to its advantages of rapidity, high sensitivity, and convenience allowing for the development of a compact, and energy-efficient platform that can facilitate real-time on-site diagnostics with accurate temperature control.

Development of a hybridization chain reaction-powered lab-on-fiber device for ultrafast point-of-care testing of circulating tuor DNA in whole blood

Circulating tumor DNA (ctDNA) demonstrates great promise in the guidance of prognostication, diagnosis, and surveillance of cancers, which highlights the need for rapid and sensitive point-of-care testing (POCT) technologies. Hybridization chain reaction (HCR)-based optical biosensors provide excellent solutions due to their prominent features. However, the requirement of a sophisticated and expensive optical readout device, relatively long detection time, and heating hold back their scalability and clinical applications. Here, an innovative HCR-powered lab-on-fiber device (HCR-LOFD) was developed for rapid on-site detection of ctDNA with high sensitivity, specificity, and reproducibility. A LOFD with a compact all-fiber optical structure was constructed for the fluorescence detection of the HCR system. Combining HCR, fluorescence energy resonant transfer, and the evanescent wave fluorescence principle, HCR-LOFD achieved the quantitative detection of KRAS G12D, the 12th amino acid from glycine (Gly) mutated aspartate (Asp) and the most common mutation of KARS, in 5 min at room temperature based on end-point detection mode or real-time fluorescence detection mode. This new assay platform was also successfully applied for the direct detection of KRAS G12D in whole blood with simple dilution. The application of HCR-LOFD not only greatly simplifies the complexity of optical readout devices and improves their scalability but also potentially serves as a sample-to-answer solution for the detection of biomarkers in limited medical resource regions.

CRISPR-assisted test for Schistosoma haematobium

Schistosomiasis is a major neglected tropical disease targeted for elimination as a public health issue by 2030, however there is an urgent need for more sensitive and specific diagnostic tests suitable to resource-limited settings. Here we developed CATSH, a CRISPR-assisted diagnostic test for Schistosoma haematobium, utilising recombinase polymerase amplification, Cas12a-targeted cleavage and portable real-time fluorescence detection. CATSH showed high analytical sensitivity, consistent detection of a single parasitic egg and specificity for urogenital Schistosoma species. Thanks to a novel CRISPR-compatible sample preparation developed using simulated urine samples containing parasitic eggs, CATSH had a sample-to-result within 2 h. The components of CATSH can be lyophilised, reducing cold chain dependence and widening access to lower and middle-income countries. This work presents a new application of CRISPR diagnostics for highly sensitive and specific detection of parasitic pathogens in remote areas and could have a significant impact on the elimination of neglected tropical diseases.