Enhanced Detection Sensitivity Research Articles

Advancements in electrochemical immunosensors towards point-of-care detection of cardiac biomarkers.

Cardiovascular disease remains the leading cause of death worldwide, with mortality rates increasing annually. This underscores the urgent need for accurate diagnostic and monitoring tools. Electrochemical detection has emerged as a promising method for swiftly and precisely measuring specific biomarkers in bodily fluids. This approach is not only cost-effective and efficient compared to traditional clinical methods, but it can also be tailored to detect individual biomarkers, which makes it particularly well-suited for point-of-care (POC) applications. The ability to conduct testing at the point of care is crucial for timely interventions and personalized disease management, empowering healthcare providers to tailor treatment plans based on real-time biomarker data. Thanks to recent advancements in nanomaterials, we've seen significant progress in electrochemical detection, leading to the development of specialized rapid immunoassay systems. These systems utilize specific antibodies to target molecules, expanding the range of detectable biomarkers. This innovation has the potential to revolutionize the diagnosis and treatment of cardiovascular diseases by enhancing detection sensitivity and specificity. Ultimately, these advancements aim to improve patient outcomes by enabling earlier diagnosis, more precise monitoring, and personalized therapeutic interventions, which will contribute to more effective management of cardiovascular health globally.

Exceptional point enhanced nanoparticle detection in deformed Reuleaux-triangle microcavity.

In this paper, we propose a deformed Reuleaux-triangle resonator (RTR) to form exceptional point (EP) which results in the detection sensitivity enhancement of nanoparticle. After introducing single nanoparticle to the deformed RTR at EP, frequency splitting obtains an enhancement of more than 6 times compared with non-deformed RTR. In addition, EP induced a result that the far field pattern of chiral mode responses significantly to external perturbation, corresponding to the change in internal chirality. Therefore, single nanoparticle with far distance of more than 4000 nm can be detected by measuring the variation of far field directional emission. Compared to traditional frequency splitting, the far field pattern produced in deformed RTR provides a cost-effective and convenient path to detect single nanoparticle at a long distance, without using tunable laser and external coupler. Our structure indicates great potential in high sensitivity sensor and label-free detector.

Lab-in-a-Vial Rapid Test for Internet of Things-Embedded Point-of-Healthcare Protein Biomarker Detection in Bodily Fluids.

Amateurs often struggle with detecting and quantifying protein biomarkers in body fluids due to the high expertise required. This study introduces a Lab-in-a-Vial (LV) rapid diagnostic platform, featuring hydrangea-like platinum nanozymes (PtNH), for rapid, accurate detection and quantification of protein biomarkers on-site within 15min. This method significantly enhances detection sensitivity for various biomarkers in body fluids, surpassing traditional methods such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays (LFA) by ≈250 to 1300 times. The LV platform uses a glass vial coated with specific bioreceptors such as antigens or antibodies, enabling rapid in vitro evaluation of disease risk from small fluid samples, similar to a personal ELISA-like point-of-care test (POCT). It overcomes challenges in on-site biomarker detection, allowing both detection and quantification through a portable wireless spectrometer for healthcare internet of things (H-IoT). The platform's effectiveness and adaptability are confirmed using IgG/IgM antibodies from SARS-CoV-2 infected patients and nuclear matrix protein (NMP22) from urothelial carcinoma (UC) patients as biomarkers. These tests demonstrated its accuracy and flexibility. This approach offers vast potential for diverse disease applications, provided that the relevant protein biomarkers in bodily fluids are identified.

In-line RNA-based microreactor direct mass spectrometry for ultrasensitive and rapid assay of terminal deoxynucleotidyl transferase activity

Terminal deoxynucleotidyl transferase (TdT), a unique template-independent DNA polymerase, plays a crucial role in the human adaptive immune system and is considered a promising biomarker for the diagnosis of various forms of acute or chronic leukemia. The accurate and sensitive detection of trace TdT is of pivotal importance to fulfill the significant medical interest in understanding its pathological functions and diagnosing TdT-related diseases. We hereby present an in-line RNA-based microreactor direct mass spectrometry (MS) method and its application for ultrasensitive, accurate, and rapid analysis of trace TdT activity in leukemic cell samples. A specially designed RNA-based microreactor is fabricated by immobilizing short RNA sequence via covalent Au–S bond on the inner surface of a capillary pre-modified with three-dimensional porous layer (PL) and Au nanoparticles (AuNPs). Utilizing this PL@Au@RNA microreactor, the signal of target TdT is conversed into reporter molecules (adenine), which exhibit a strong MS response. This conversion process enables efficient signal amplification and enhances detection sensitivity. The outlet end of the PL@Au@RNA microreactor is deliberately crafted into a porous tip, serving as an electrospray ionization (ESI) interface to directly couple to ESI-MS in-line. This design facilitates the direct transmission of the generated signaling molecules into the MS system, eliminating the need for laborious sample treatment procedures. By implementing this RNA-based microreactor in direct MS analysis, we have achieved remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10−9 U, surpassing other reported methods in literature by three to four orders of magnitude. Furthermore, each assay requires a minimal sample volume of merely 10 nL. This method has successfully demonstrated its application in accurately and efficiently detecting TdT activity in leukemia cells, and its detection results are consistent with those obtained by ELISA kits.

Magneto-Optical Ceramics with High Transparency for Highly Sensitive Magnetometer via Quantum Weak Measurement.

Sensitive magnetometer technology is desirable for biomagnetic field detection and geomagnetic field measuring. Signal amplification materials such as magneto-optical crystals or ceramics are crucial for enhancing detection sensitivity, but severe optical scattering and low Verdet constant further limit its application. To develop high-sensitivity magnetometers for quantum weak measurement schemes, we have conducted investigations on the powder calcining dynamics and prepared a series of high-optical-quality (Ho/Dy)2Zr2O7 transparent ceramic samples. The Verdet constant of magneto-optical materials was measured across a continuous wavelength spectrum, exhibiting a peak at 283 ± 5 rad/(T·m). We further established an electron transition mechanism to elucidate the exceptional magneto-optical attributes of dysprosium. In addition, samples demonstrated superior performance in weak-value amplification, reaching a low detectable magnetic field threshold of 3.5 × 10-8 T and continuously worked over 6 h with high stability. Our work developed a highly sensitive magnetometer using optimized magneto-optical ceramics and provided guidance on design, fabrication, and application for magneto-optical ceramics in quantum weak measurement.

Label-free multi-line immunochromatographic sensor based on TCBPE for broad-spectrum detection Salmonella in food

BackgroundSalmonella, a foodborne pathogen poses significant threats to food safety and human health. Immunochromatographic (ICTS) sensors have gained popularity in the field of food safety due to their convenience, speed, and cost-effectiveness. However, most existing ICTS sensors rely on antibody sandwich structures which are limited by their dependence on high-quality paired antibodies and restricted sensitivity. For the first time, we combined multi-line ICTS strips with fluorescent bacterial probes to develop a label-free multi-line immunochromatographic sensor capable of detecting broad-spectrum Salmonella. Salmonella was labeled with the aggregation-induced luminescence material TCBPE, resulting in its transformation into a green fluorescent probe. ResultsUsing this sensor, we successfully detected Salmonella typhimurium within the concentration range of 104 to 108 CFU/mL with a visual detection limit of 6.0×104 CFU/mL. Compared to single-line sensors, our multi-line sensor exhibited significantly improved fluorescence intensity resulting in enhanced detection sensitivity by 50%. Furthermore, our developed multi-line ICTS sensor demonstrated successful detection of 18 different strains of Salmonella without any cross-reaction observed with 5 common foodborne pathogens tested. The applicability and reliability were validated using milk samples, cabbage juice samples as well and drinking water samples suggesting its potential for rapid and accurate detection of Salmonella in real-world scenarios across both the food industry and clinical settings. SignificanceIn this experiment, we developed a TCBPE-based multiline immunochromatographic sensor. Specifically, Salmonella was labeled with the aggregation-induced luminescence material TCBPE, resulting in its transformation into a green fluorescent probe. Through the multi-line analysis system, the detection sensitivity and accuracy of the sensor are improved. In brief, the sensor does not require complex antibody labeling and paired antibodies, and only one antibody is needed to complete the detection process.

Enhanced detection sensitivity of X-ray detectors via CdSe nanoplatelet aspect ratio control

This study investigated the use of inorganic cadmium selenide nanoplatelets (CdSe NPLs) to enhance the performance of X-ray detectors. The active layer of the detector comprised a bulk heterojunction composed of PCDTBT:PC71BM: CdSe NPLs with three different aspect ratios (ARs = 3:1, 1:1, and 6:1). The CdSe NPLs, synthesized with a thickness of five monolayers, were controlled by adjusting the molar mass ratios of Cd(OAc)2 and Cd(OAc)2∙2 H2O. Each detector was characterized by analyzing its series resistance, collection irradiation, dark current density, detection sensitivity, charge mobility, and defect density via the collected charges. The detector containing CdSe NPLs with an aspect ratio of 1:1 demonstrated the most favorable performance, exhibiting improved carrier generation and transport properties, resulting in a 23.36 % increase in sensitivity compared with the detector without CdSe NPLs. Through experimentation, the optimal content of CdSe NPLs in the active layer was determined to be 1 wt%, yielding the highest sensitivity of 3.96 × 103 μC∙Gy−1∙cm−2. This study underscores the potential of CdSe NPLs as promising materials for X-ray detector applications.

Highly efficient and sensitive detection of tetracycline in environmental water: Insights into the synergistic mechanism of biomass-derived carbon dots and N-methyl pyrrolidone solvent

The tetracycline (TC) residue in water environment has caused serious public safety issue. Thus, efficient sensing of TC is highly desirable for environmental protection. Herein, biomass-derived nitrogen-doped carbon dots (N-CDs) synthesized from natural Ophiopogon japonicus f. nanus (O. japonicus) were used for TC detection. The unique solvent synergism efficiently enhanced detection sensitivity, and the detailed sensing mechanism was deeply investigated. The blue fluorescence of N-CDs was quenched by TC via static quenching and inner filter effect. Moreover, the enhancement of green fluorescence from deprotonated TC was firstly proposed and sufficiently verified. The solvent effect of N-methyl pyrrolidone (NMP) and the fluorescence resonance energy transfer (FRET) with N-CDs achieved an instantaneous enhancement of the green emission by 64-fold. Accordingly, a ratiometric fluorescence method was constructed for rapid and sensitive sensing of TC with a low detection limit of 6.3 nM within 60 s. The synergistic effect of N-CDs and solvent assistance significantly improved the sensitivity by 7-fold compared to that in water. Remarkably, the biomass-derived N-CDs displayed low cost, good solubility, and desired stability. The deep insights into the synergism with solvent can provide prospects for the utilization of biomass-based materials and broaden the development of advanced sensors with promising applications.

Internal flow in sessile droplets induced by substrate oscillation: towards enhanced mixing and mass transfer in microfluidic systems

The introduction of flows within sessile droplets is highly effective for many lab-on-a-chip chemical and biomedical applications. However, generating such flows is difficult due to the typically small droplet volumes. Here, we present a simple, non-contact strategy to generate internal flows in sessile droplets for enhancing mixing and mass transport. The flows are driven by actuating a rigid substrate into oscillation with certain amplitude distributions without relying on the resonance of the droplet itself. Substrate oscillation characteristics and corresponding flow patterns are documented herein. Mixing indices and mass transfer coefficients of sessile droplets on the substrate surface are measured using optical and electrochemical methods. They demonstrate complete mixing within the droplets in 1.35 s and increases in mass transfer rates of more than seven times static values. Proof of concept was conducted with experiments of silver nanoparticle synthesis and with heavy metal ion sensing employing the sessile droplet as a microreactor for synthesis and an electrochemical cell for sensing. The degrees of enhancement of synthesis efficiency and detection sensitivity attributed to the internal flows are experimentally documented.

All-in-one platform: Versatile, Easy, and User-friendly System (VEUS) based on automated and expert-independent antibody immobilization and immunoassay by utilizing customized movement of magnetic particles.

The ELISA is the most worldwide method for immunoassay. However, the ELISA is losing ground due to low reproducibility of manual experimental processes in both R&D and IVD areas. An automated platform is a good solution, but there are still limitations owning to extremely high cost and requiring large space to set up especially for a small size laboratory. Here, we present a novel all-in-one platform called "VEUS" settable on the laboratory table that offers comprehensive automation of the entire multiplex immunoassay process by exploiting antibody conjugated magnetic particles, quality control and then immunoanalytical reaction, thereby enhancing detection sensitivity and high reproducibility. As a proof of concept, the system exhibits a sensitive LOD of 0.6 and 3.1 pg mL-1 within 1 h run, comparable precision that of molecular diagnostic systems based on PCR method, enabling rapid multiplex diagnosis of Influenza A, Influenza B, and COVID-19 viruses with similar symptoms. Through automation by the all-in-one system, it can be used by novice users, something innovative for immunoassays, relying heavily on user experience. Furthermore, it can contribute to streamline entire immunoassay processes of diverse biomarkers with high reproducibility and convenience in laboratories.

Determination of fatty acid composition after saponification of common oil pharmaceutical excipients by supercritical fluid-evaporative light scattering method and its application in oil identification

Oils and fats are commonly used in the pharmaceutical industry as solvents, emulsifiers, wetting agents, and dispersants, and are an important category of pharmaceutical excipients. Fatty acids with unique compositions are important components of oil pharmaceutical excipients. The Chinese Pharmacopoeia provides clear descriptions of the fatty acid types and limits suitable for individual oil pharmaceutical excipient. An unqualified fatty acid composition or content may indicate adulteration or deterioration. The fatty acid composition, as a key indicator for the identification and adulteration evaluation of oil pharmaceutical excipients, can directly affect the quality and safety of oil pharmaceutical excipients and preparations. Gas chromatography is the most widely used technique for fatty acid analysis, but it generally requires derivatization, which affects quantitative accuracy. Supercritical fluid chromatography (SFC), an environmentally friendly technique with excellent separation capability, offers an efficient method for detecting fatty acids without derivatization. Unlike other chromatographic methods, SFC does not use nonvolatile solvents (e. g., water) as the mobile phase, rendering it compatible with an evaporative light-scattering detector (ELSD) for enhanced detection sensitivity. However, the fatty acids in oil pharmaceutical excipients exist in the free and bound forms, and the low content of free fatty acids in these oil pharmaceutical excipients not only poses challenges for their detection but also complicates the determination of characteristic fatty acid compositions and contents. Moreover, the compositions and ratios of fatty acids are influenced by environmental factors, leading to interconversion between their two forms. In this context, saponification provides a simpler and faster alternative to derivatization. Saponification degrades oils and fats by utilizing the reaction between esters and an alkaline solution, ultimately releasing the corresponding fatty acids. Because this method is more cost effective than derivatization, it is a suitable pretreatment method for the detection of fatty acids in oil pharmaceutical excipients using the SFC-ELSD approach. In this study, we employed SFC-ELSD to simultaneously determine six fatty acids, namely, myristic acid, palmitic acid, stearic acid, arachidic acid, docosanoic acid, and lignoceric acid, in oil pharmaceutical excipients. Saponification of the oil pharmaceutical excipients using sodium hydroxide methanol solution effectively avoided the bias in the determination of fatty acid species and contents caused by the interconversion of fatty acids and esters. The separation of the six fatty acids was achieved within 12 min, with good linearity within their respective mass concentration ranges. The limits of detection and quantification were 5-10 mg/L and 10-25 mg/L, respectively, and the spiked recoveries were 80.93%-111.66%. The method proved to be sensitive, reproducible, and stable, adequately meeting requirements for the analysis of fatty acids in oil pharmaceutical excipients. Finally, the analytical method was successfully applied to the determination of six fatty acids in five types of oil pharmaceutical excipients, namely, corn oil, soybean oil, coconut oil, olive oil, and peanut oil. It can be combined with principal component analysis to accurately differentiate different types of oil pharmaceutical excipients, providing technical support for the rapid identification and quality control of oil pharmaceutical excipients. Thus, the proposed method may potentially be applied to the analysis of complex systems adulterated with oil pharmaceutical excipients.

Ultrasensitive Detection of Tumor Suppressor Gene Methylation by Piezoelectric Sensing Based on Enrichment of Transcription Activator-Like Effectors.

The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.

Applications of Metals and Metal Compounds in Improving the Sensitivity of Microfluidic Biosensors - A Review.

The enhancement of detection sensitivity in microfluidic sensors has been a continuously explored field. Initially, many strategies for sensitivity improvement involved introducing enzyme cascade reactions, but enzyme-based reactions posed challenges in terms of cost, stability, and storage. Therefore, there is an urgent need to explore enzyme-free cascade amplification methods, which are crucial for expanding the application range and improving detection stability. Metal or metal compound nanomaterials have gained great attention in the exploitation of microfluidic sensors due to their ease of preparation, storage, and lower cost. The unique physical properties of metallic nanomaterials, including surface plasmon resonance, surface-enhanced Raman scattering, metal-enhanced fluorescence, and surface-enhanced infrared absorption, contribute significantly to enhancing detection capabilities. The metal-based catalytic nanomaterials, exemplified by Fe3O4 nanoparticles and metal-organic frameworks, are considered viable alternatives to biological enzymes due to their excellent performance. Herein, we provide a detailed overview of the applications of metals and metal compounds in improving the sensitivity of microfluidic biosensors. This review not only highlights the current developments but also critically analyzes the challenges encountered in this field. Furthermore, it outlines potential directions for future research, contributing to the ongoing development of microfluidic biosensors with improved detection sensitivity.

Passive Wireless Partial Discharge Sensors with Multiple Resonances.

Partial discharge (PD) is the dominant insulating defect in Gas-Insulated Switchgear (GIS). The existing detection methods are mainly divided into built-in wire-connected disk antennas with destructive drilling and external ultra-high frequency antennas with poor anti-interference ability. This research introduces a passive wireless PD sensor implanted inside GIS on the observation window. The sensor is implemented by a sheeting branch-inductor with multiple resonances which is able to enhance detection sensitivity. A coaxially aligned readout circuit, positioned outside the GIS, interrogates the PD sensor to wirelessly obtain the PD signal. The proposed sensing scheme improves signal-to-noise ratio and ensures minimal disruption to the electric field distribution inside GIS. An experimental setup was established in a controlled laboratory environment to benchmark the multi-resonant sensor against the commercial UHF sensor. A 2.5-times enhancement of signal strength was observed. Since our sensor was implanted inside the GIS, a high signal-to-noise ratio (68.82 dB) was obtained. Moreover, we constructed a wireless calibration test to investigate the accuracy of the proposed sensor. The precision of the signal test was as high as 0.72 pC. The pulse phase distribution information was collected to demonstrate a phase-resolved partial discharge (PRPD) pattern. The experiment results validate the effectiveness of the proposed method and demonstrate excellent performance in PD detection.

Phosphorylation-amplified synchronized droplet microfluidics sensitizes bacterial growth kinetic real-time monitoring

The necessity for rapid and accurate bacterial growth monitoring is imperative across various domains, including healthcare and environmental safety. We introduce the self-synchronized droplet-amplified electrical screening cytometry (SYNC) system, a novel meld of droplet microfluidics and electrochemical amplification tailored for precise bacterial growth kinetic monitoring. SYNC encapsulates single bacteria in picolitre droplets, enabling real-time, fluorescence-free electrochemical monitoring. A specially devised phosphorylation-amplified culture medium translates bacterial metabolic activity into discernible electrical impedance changes. The dual-channel design and a rail-based structure in SYNC facilitate parallel screening and self-synchronization of droplets, addressing the limitations of conventional impedance cytometry. SYNC showcases a 5-fold enhancement in detection sensitivity and reduces 50% of the detection time compared to traditional approaches. Notably, SYNC is pioneering in providing exact initial bacterial concentrations, achieve to 104 bacteria/ml, a capability unmatched by existing real-time techniques measuring electrochemical variations. Along with its robust performance, this earmarks SYNC as a powerful tool for applications such as antibiotic susceptibility testing, food quality monitoring, and real-time water bacteria monitoring, paving the way for enhanced microbial process management and infection control.

A simple and green offline-online capillary electrophoresis stacking strategy for the simultaneous determination of hydrophobic compounds in complicated samples using sodium dodecyl sulfate as the solubilizer and pseudophase

BackgroundCapillary electrophoresis is a powerful analytical method featured with high separation efficiency, minimal sample requirements, and reduced organic solvents consumption. However, its low sensitivity hinders its wide application in determination of trace analytes especially for the weakly ionized hydrophobic compounds. Offline and Online capillary electrophoresis stacking methods are more favored to enhance detection sensitivity of analytes. The determination of two sesquiterpenes and an alkaloid from the dried root of Lindera aggregata merged as an example for developing a simple, sensitive and green method for the simultaneous determination of two hydrophobic compounds in complicated matrix samples. ResultsAn offline-online capillary electrophoresis stacking strategy by integrating micro matrix solid phase dispersion with field-amplified sample stacking and micelle to cyclodextrin stacking has been developed for the simultaneous determination of dehydrocostus lactone, linderane, norisoboldine in complex matrices. The optimized parameters were set at 65 mM sodium dihydrogen phosphate, 35 % methanol, 180 s for sample injection and 210 s for cyclodextrin injection, 20 mM sodium dodecyl sulfate of sample matrix for online stacking; 1:1 sample to MCM-48, 180 s grinding time, and 1000 μL of 20 mM sodium dodecyl sulfate elution for offline procedure. Under the optimum conditions, the method showed good linearity with correlation coefficients (R2 ≥ 0.9927), low limits of detection within the range of 25–50 ng mL−1, satisfactory repeatability and reproducibility below 3.98 %, and acceptable recoveries between 94 % and 97 %. The developed method was successfully applied to two real samples, the root of L. aggregata and rat feces. SignificanceSodium dodecyl sulfate is firstly used as an eluent in micro matrix solid phase dispersion and plays a dual role throughout the analytical procedure, including extraction solvent in sample preparation and micelle pseudophase during online stacking. It brings great procedure convenience to the method. The sensitivity of this method can improve up to 1283-folds compared with the normal mode. Moreover, the overall strategy indicates satisfied green potential evaluated by greenness assessment tools.

Special RCA based sensitive point‐of‐care detection of HPV mRNA for cervical cancer screening

AbstractDeveloping the sensitive point‐of‐care testing (POCT) of oncogenic nucleic acids from human papillomavirus (HPV) infection is essential in preventing cervical cancer, especially in resource‐limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT via nucleic acid‐based aggregation under isothermal conditions. However, the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity. Here, a minimum secondary structured RCA technique (MSS‐RCA) is developed by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions. The amplification efficiency of MSS‐RCA could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS‐RCA, HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 fm (50 zmol in 10 µL). This integrated MSS‐RCA‐PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS‐RCA‐PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA

BackgroundCRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. ResultsHere, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3′ end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. SignificanceThe one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

Surface-active agent enhanced FRET effect Cu-doped NH2-MIL-88(Fe) for highly sensitive detection of 3-nitro-L-tyrosine

In this study, Cu-doped NH2-MIL-88(Fe) metal–organic frameworks (MOF) were synthesized via a one-step method. Characterization techniques such as XPS, XRD and FTIR confirmed the successful incorporation of Cu2+ into NH2-MIL-88(Fe), naming this MOF as NH2-MIL-88(Fe)@Cu2+. This MOF was employed to develop a highly sensitive fluorescence sensing platform for detecting 3-nitro-L-tyrosine(3-NT). The potential for fluorescence resonance energy transfer (FRET) was suggested by the spectral overlap between NH2-MIL-88(Fe)@Cu2+'s emission and 3-NT's UV absorption. To augment this effect, cationic surfactant hexadecyltrimethylammonium bromide (CTAB), which self-assembled into nanostructured microspheres above its critical micelle concentration, was utilized. The charged surface of these microspheres, formed by the self-assembly of CTAB, is bound to the MOF surface through electrostatic force and simultaneously attracts 3-NT. Adjusting the solution's pH strengthened the interaction between NH2-MIL-88(Fe)@Cu2+ and 3-NT, thereby enhancing their mutual FRET interaction. Experimental results indicated that CTAB's introduction markedly improved the FRET effects, potentially converting a weak FRET into a strong one and enhancing detection sensitivity and accuracy. Under optimal conditions, NH2-MIL-88(Fe)@Cu2+ detected 3-NT within 0–30 μM range, with a limit of detection (LOD, S/N = 3) of 41.1 nM. Finally, the applicability of the sensor is tested by calibrating measurements in fetal bovine serum samples, achieving good performance in terms of sensitivity, selectivity and reproducibility. This research provides a method for efficient and highly sensitive 3-NT detection and insights into the FRET effect between MOF and target molecules, likely advancing related fields and inspiring future fluorescence sensor designs.

A novel entropy-driven dual-output mode integrated with DNAzyme for enhanced microRNA detection

Accurate microRNA (miRNA) detection is pivotal in the diagnosis and monitoring of cancer. Entropy-driven catalysis (EDC) has attracted widespread attention as an enzyme-free, isothermal technique for miRNA detection owing to its inherent simplicity and reliability. However, conventional EDC is a single-output mode, limiting the efficiency of signal amplification. In this study, a novel EDC dual-output mode was employed in conjunction with DNAzyme, resulting in the development of an EDC dual-end DNAzyme (EDC-DED) approach for highly sensitive miRNA detection. In this system, miRNA-21 initiated the EDC reaction, producing a large amount of catalytically active dual-end Mg2+-dependent DNAzyme. The DNAzyme further cleaved the reporter cyclically, generating a notably amplified fluorescence signal. The proposed method achieved a low detection limit of 2 pM. Compared with the traditional EDC single-end DNAzyme (EDC-SED) strategy, the present method exhibited superior amplification efficiency, enhancing detection sensitivity by approximately 46.5-fold. Furthermore, this platform demonstrated ideal specificity, satisfactory reproducibility and acceptable detection capabilities in clinical serum samples. Therefore, the straightforward and convenient strategy is a potential tool for miRNA analysis, which may provide a new perspective for biological analysis and clinical application.