Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads.

Nucleic acids are important biomarkers in cancer and viral diseases. However, their ultralow concentration in biological/clinical samples makes direct target detection challenging, because it leads to slow hybridization kinetics with the probe and its insufficient signal-to-noise ratio. Therefore, RNA target detection is done by molecular (target) amplification, notably by RT-PCR, which is a tedious multistep method that includes nucleic acid extraction and reverse transcription. Here, a direct method based on ultrabright dye-loaded polymeric nanoparticles in a sandwich-like hybridization assay with magnetic beads is reported. The ultrabright DNA-functionalized nanoparticle, equivalent to ≈10 000 strongly emissive rhodamine dyes, is hybridized with the magnetic bead to the RNA target, providing the signal amplification for the detection. This concept (magneto-fluorescent sandwich) enables high-throughput detection of DNA and RNA sequences of varied lengths from 48 to 1362 nt with the limit of detection down to 0.3 fm using a plate reader (15 zeptomoles), among the best reported for optical sandwich assays. Moreover, it allows semi-quantitative detection of SARS-CoV-2 viral RNA directly in clinical samples without a dedicated RNA extraction step. The developed technology, combining ultrabright nanoparticles with magnetic beads, addresses fundamental challenges in RNA detection; it is expected to accelerate molecular diagnostics of diseases.

Enhancing the activation of T cells through anti-CD3/CD28 magnetic beads by adjusting the antibody ratio.

The utilization of anti-CD3/CD28 magnetic beads for T cell expansion in vitro has been investigated for adoptive cell transfer therapy. However, the impact of the CD3/CD28 antibody ratio on T cell differentiation and function remains incompletely elucidated. This study seeks to address this knowledge gap. To begin with, CD3 antibodies with a relatively low avidity for Jurkat cells (Kd = 13.55 nM) and CD28 antibodies with a relatively high avidity (Kd = 5.79 nM) were prepared. Afterwards, anti-CD3/CD28 antibodies with different mass ratios were attached to magnetic beads to examine the impacts of different antibody ratios on T cell capture, and proliferation. The research demonstrated that the most significant expansion of T cells was stimulated by the anti-CD3/CD28 magnetic beads with a mass ratio of 2:1 for CD3 antibodies and CD28 antibodies. Moreover, CD25 and PD1 expression of expanded T cells increased and then decreased, with lower CD25 and PD1 expression in the later stages of expansion indicating that T cells were not depleted. These T cells, which are massively expanded in vitro and have excellent expansion potential, can be infused back into the patient to treat tumor patients. This study shows that altering the ratio of anti-CD3/CD28 antibodies can control the strength of T cell stimulation, thereby leading to the improvement of T cell activation. This discovery can be utilized as a guide for the creation of other T cell stimulation approaches, which is beneficial for the further development of tumor immunotherapy technology.

Periodic magnetic modulation enhanced electrochemical analysis for highly sensitive determination of genomic DNA methylation.

DNA methylation aberrations have a strong correlation with cancer in early detection, diagnosis, and prognosis, which make them possible candidate biomarkers. Electrochemical biosensors offer rapid protocols for detecting DNA methylation status with minimal pretreatment of samples. However, the inevitable presence of background current in the time domain, including electrochemical noise and variations, limits the detection performance of these biosensors, especially for low concentration analytes. Here, we propose an ultrasensitive frequency-domain electrochemical analysis strategy to effectively separate the weak signals from background current. To achieve this, we employed periodic magnetic field modulation of magnetic beads (MBs) on and off the electrode surface to generate a periodic electrochemical signal for subsequent frequency-domain analysis. By capturing labeled MBs with as low as 0.5 pg of DNA, we successfully demonstrated a highly sensitive electrochemical method for determination of genome-wide DNA methylation levels. We also validated the effectiveness of this methodology using DNA samples extracted from three types of hepatocellular carcinoma (HCC) cell lines. The results revealed varying genomic methylation levels among different HCC cell lines, indicating the potential application of this approach for early-stage cancer detection in terms of DNA methylation status.

Visual fluorescence detection of Listeria monocytogenes with CRISPR-Cas12a aptasensor.

Listeria monocytogenes (L. monocytogenes) is a prevalent food-borne pathogen that can cause listeriosis, which manifests as meningitis and other symptoms, potentially leading to fatal outcomes in severe cases. In this study, we developed an aptasensor utilizing carboxylated magnetic beads and Cas12a to detect L. monocytogenes. In the absence of L. monocytogenes, the aptamer maintains its spatial configuration, keeping the double-stranded DNA attached and preventing the release of a startup template and activation of Cas12a's trans-cleavage capability. Conversely, in the presence of L. monocytogenes, the aptamer undergoes a conformational change, releasing the double-stranded DNA to serve as a startup template, thereby activating the trans-cleavage capability of Cas12a. Consequently, as the concentration of L. monocytogenes increases, the observable brightness in a blue light gel cutter intensifies, leading to a rise in fluorescence intensity difference compared to the control. This Cas12a aptasensor demonstrates excellent sensitivity towards L. monocytogenes, with a lowest detection limit (LOD) of 57.15 CFU/mL and a linear range of 4×102 to 4×107 CFU/mL (R2=0.9858). Notably, the proposed Cas12a aptasensor exhibited outstanding selectivity and recovery in beef samples, and could be employed for precise monitoring. This Cas12a aptasensor not only provides a novel fluorescent and visual rapid detection method for L. monocytogenes but also offers simplicity, speed, and stability compared to previous detection methods. Furthermore, it is suitable for on-site detection of beef samples.

Accidental ingestion of multiple magnetic beads by children and their impact on the gastrointestinal tract: a single-center study.

OBJECTIVE: In this study, we aimed to enhance the treatment protocols and help understand the harm caused by the accidental ingestion of magnetic beads by children. METHODS: Data were collected from 72 children with multiple gastrointestinal perforations or gastrointestinal obstructions. The 72 pediatric patients were divided into a perforation and a non-perforation group. The data collected for the analysis included the gender, age, medical history, place of residence (rural or urban), and symptoms along with the educational background of the caregiver, the location and quantity of any foreign bodies discovered during the procedure, whether perforation was confirmed during the procedure, and the number of times magnetic beads had been accidentally ingested. RESULTS: The accuracy rate of preoperative gastrointestinal perforation diagnosis via ultrasound was 71%, while that of the upright abdominal X-ray method was only 46%. In terms of symptoms, the risk of perforation was 13.844 and 12.703 times greater in pediatric patients who experienced vomiting and abdominal pain with vomiting and abdominal distension, respectively, compared to patients in an asymptomatic state. There were no statistical differences between the perforation and the non-perforation groups in terms of age, gender, medical history, and the number of magnetic beads ingested (P > 0.05); however, there were statistical differences in terms of white blood cell count (P = 0.048) and c-reactive protein levels (P = 0.033). A total of 56% of cases underwent a laparotomy along with perforation repair and 19% underwent gastroscopy along with laparotomy. All pediatric patients recovered without complications following surgery. CONCLUSION: Abdominal ultrasonography and/or upright abdominal X-ray analyses should be carried out as soon as possible in case of suspicion of accidental ingestion of magnetic beads by children. In most cases, immediate surgical intervention is required. Given the serious consequences of ingesting this type of foreign body, it is essential to inform parents and/or caregivers about the importance of preventing young children from using such products.

Examining the efficiency of porcine gastric mucin-coated magnetic beads in extraction of noroviruses from frozen berries.

Human norovirus is the leading cause of foodborne gastroenteritis worldwide. Due to the low infectious dose of noroviruses, sensitive methodologies are required to detect and characterize small numbers of viral particles that are found in contaminated foods. The ISO 15216 method, which is internationally recognized for detection of foodborne viruses from high-risk food commodities, is based on viral precipitation, followed by RNA extraction and identification of the viral genome by RT-PCR. Although the ISO 15216 method is efficient, it is time consuming and tedious, does not report on the viral infectivity, and is sensitive to the presence of RT-PCR inhibitors. Norovirus capture by the porcine gastric mucin conjugated magnetic beads (PGM-MB) was developed as an alternative virus recovery method. It relies on the integrity of the viral capsid being able to bind to PGM. PGM contains a variety of histo-blood group antigens (HBGAs) that act as norovirus receptors. Therefore, the PGM-MB method allows for extraction of noroviruses, with potentially intact viral capsids, from complex food matrices. The viral genome can then be released through heat-shock of the captured virus. For this reason, we performed a parallel comparison between the ISO 15216 method and the PGM-MB method in isolation and quantification of noroviruses from frozen raspberries. We have demonstrated that the efficiency of the PGM-MB method in extraction of murine norovirus (MNV) and human norovirus GII.4 from raspberries is equal or better than the ISO 15216 method, while the PGM-MB has fewer steps and shorter turnaround time. Moreover, the PGM-MB method is more efficient in removing the inhibitors prior to RT-PCR analysis.

Bi-functionality of glyoxal caged nucleic acid coupled with CRISPR/Cas12a system for Hg2+ determination.

A highly sensitive and selective fluorescence method has been conducted for the detection of Hg2+ based on aminophenylboronic acid-modified carboxyl magnetic beads (CMB@APBA) and CRISPR/Cas12a system mediated by glyoxal caged nucleic acid (gcDNA). As a bi-functional DNA linker, gcDNA offers advantages of simultaneous recognition by boronic acid and complementary DNA/RNA. Under acidic condition, gcDNA can be immobilized on CMB@APBA through the formation of borate ester bond. The formed boric acid-esterified gcDNA can further bind with complementary CRISPR RNA through A-T base pairing to activate Cas12a with kcat/Km ratio of 3.4 × 107 s-1 M-1, allowing for amplified signal. Hg2+ can specifically combine with CMB@APBA, resulting in the release of gcDNA from CMB@APBA and the following inhibition on the activation of CRISPR/Cas12a system around magnetic bead. Under optimal conditions, the method exhibits a linear range from 20 to 250 nM, with a detection limit of 2.72 nM. The proposed method can detect Hg2+ in milk and tea beverages, providing a great significance for on-site monitoring of Hg2+ contamination in food.

Recent Trends and Innovations in Bead-Based Biosensors for Cancer Detection.

Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as promising diagnostic platforms based on wide-ranging cancer biomarkers owing to the versatility, high sensitivity, and flexibility to perform the multiplexing of beads. This comprehensive review highlights recent trends and innovations in the development of bead-based biosensors for cancer-biomarker detection. We introduce various types of bead-based biosensors such as optical, electrochemical, and magnetic biosensors, along with their respective advantages and limitations. Moreover, the review summarizes the latest advancements, including fabrication techniques, signal-amplification strategies, and integration with microfluidics and nanotechnology. Additionally, the challenges and future perspectives in the field of bead-based biosensors for cancer-biomarker detection are discussed. Understanding these innovations in bead-based biosensors can greatly contribute to improvements in cancer diagnostics, thereby facilitating early detection and personalized treatments.

Automated Mass Photometry of Adeno-Associated Virus Vectors from Crude Cell Extracts.

Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.

Efficient AAV9 Purification Using a Single-Step AAV9 Magnetic Affinity Beads Isolation.

Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results. In this study, we explore a novel purification strategy utilizing Dynabeads™ CaptureSelect™ magnetic beads. The AAV9 magnetic beads capture AAV9 with high specificity and recovery between 70 and 90%, whereas the AAVX magnetic beads did not bind to the AAV9. Through continuous interaction with AAVs in solution, these beads offer enhanced clearance of genomic DNA and plasmids even in the absence of endonuclease. The beads could be regenerated at least eight times, and the used beads could be stored for up to six months and reused without a significant reduction in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.

"Necklace of magnets" in the abdomen: an unusual case with "multiple" bowel perforations.

Magnetic foreign body ingestion poses a threat especially if more than one is ingested. If consumed alone, small magnetic foreign bodies are likely to pass without significant event; however, when multiple magnets are ingested, they can be attracted to each other through the intestinal wall, which may lead to serious consequences and complications, including bowel perforation, obstruction, peritonitis, and death. We report a case of a 2-years male child patient presented with multiple small round magnetic beads ingestion from a magnetic pendant that appeared like a necklace pearl after conglomeration on abdominal radiograph. On exploration, we found multiple perforations involving ileum, cecum, and transverse colon, with multiple conglomerated beads extruding from the perforation sites.

Separating the chaff from the wheat: antibody-based removal of DNA-fragmented sperm.

STUDY QUESTION: Is it possible to remove sperm with damaged DNA from a semen sample? SUMMARY ANSWER: By using immunomagnetic cell sorting that targets the sperm head-bound epididymal sperm-binding protein 1 (ELSPBP1), it was possible to produce an ELSPBP1(-) sperm fraction characterized by consistently lower levels of sperm DNA fragmentation (SDF). WHAT IS KNOWN ALREADY: In bovines, ELSPBP1 is bound to dead spermatozoa. Human ejaculates with high SDF have increased detected levels of sperm ELSPBP1 when compared to ejaculates with low native SDF. STUDY DESIGN, SIZE, DURATION: We recruited 267 patients who were referred to the clinic for conjugal infertility. After applying exclusion criteria, such as fever within 90 days of the study, history of systemic diseases, alterations or surgical interventions to the genital tract and use of cigarette or drugs, a total of 133 patients were included. A total of 52 samples were used for the evaluation of sperm ELSPBP1 levels (Sub-study 1), 41 samples for determination of ELSPBP1 location in human sperm (Sub-study 2), and 40 samples for immunomagnetic cell sorting targeting ELSPBP1, to produce ELSPBP1(-) (without ELSPBP1) and ELSPBP1(+) (with ELSPBP1) fractions (Sub-study 3). Samples were collected between July 2016 and September 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In Sub-study 1, sperm ELSPBP1 levels were assessed by western blotting. For Sub-study 2, ELSPBP1 was localized in sperm by immunocytochemistry. Finally, for Sub-study 3, sperm were selected based on incubation of semen samples with antibody-coated magnetic microspheres targeting ELSPBP1. Two fractions were produced (with or without ELSPBP1), and these sub-populations were submitted to an alkaline Comet assay for determination of SDF. MAIN RESULTS AND THE ROLE OF CHANCE: Men with high SDF presented higher sperm ELSPBP1 levels when compared to the control group (low SDF), while no difference between groups was observed in seminal plasma. ELSPBP1 was located in the head region of human sperm. The ELSPBP1(+) fractions presented high and variable levels of SDF, while their paired ELSPBP(-) fractions presented consistently low SDF. LIMITATIONS, REASONS FOR CAUTION: This work did not validate the levels of ELSPBP1 in other functional alterations of sperm, such as acrosome integrity or mitochondrial activity. Moreover, this is still a pre-clinical study, intended to demonstrate proof-of-concept that ELSPBP1 selects sperm with low DNA fragmentation; further investigation is warranted to demonstrate safety for use in ART. Sperm fractions were not assessed for sperm vitality. A clinical trial is still necessary for these findings to be extrapolated to outcomes in ART. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that ELSPBP1 is associated with sperm with higher levels of DNA fragmentation. The finding that the sperm membrane can reflect alterations in DNA integrity could give rise to a novel molecular method for sperm preparation prior to use of assisted reproductive procedures. Moreover, the detection of sperm-bound ELSPBP1 could serve as an indirect method for the determination of DNA fragmentation. STUDY FUNDING/COMPETING INTEREST(S): L.B.B. was a recipient of a Ph.D. scholarship from the Sao Paulo Research Foundation-FAPESP (process number 2016/05487-3). R.P.B. is a recipient of a Scientific Productivity scholarship from the Brazilian National Council for Scientific and Technological Development-CNPq (process number 306705/2017-6). The authors have no conflict of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth.

Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A-coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.

Direct capture and amplification of nucleic acids using a universal, elution-free magnetic bead-based method for rapid pathogen detection in multiple types of biological samples.

Nucleic acid amplification tests (NAATs) have become an attractive approach for pathogen detection, and obtaining high-quality nucleic acid extracts from biological samples plays a critical role in ensuring accurate NAATs. In this work, we established an elution-free magnetic bead (MB)-based method by introducing polyethylene-polypropylene glycol (PEPPG) F68 in lysis buffer and using NaOH solution instead of alcohols as the washing buffer for rapid nucleic acid extraction from multiple types of biological samples, including nasopharyngeal swabs, serum, milk, and pork, which bypassed the nucleic acid elution step and allowed the nucleic acid/MB composite to be directly used as the template for amplification reactions. The entire extraction process was able to be completed in approximately 7 min. Even though the nucleic acid/MB composite could not be used for quantitative real-time PCR (qPCR) assays, this elution-free MB-based method significantly improved the sensitivity of the loop-mediated isothermal amplification (LAMP) assay. The sensitivity of the quantitative real-time LAMP (qLAMP) assays combined with this elution-free MB-based method showed an improvement of one to three orders of magnitude compared with qLAMP or qPCR assays combined with the traditional MB-based method. In addition to manual operation, like the traditional MB-based method, this universal, rapid, and facile nucleic acid extraction method also has potential for integration into automated robotic processing, making it particularly suitable for the establishment of an analysis platform for ultrafast and sensitive pathogen detection in various biological samples both in centralized laboratories and at remote sites.

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen.

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 µm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.

A strand displacement signal amplification-assisted fluorescence assay for sensitive detection of microRNA.

MicroRNA is a vital biomarker because of its abnormal expression in the emergence and development of diseases, especially in cancers. Herein, a label-free fluorescent sensing platform is proposed for detecting microRNA-21, coupled with the cascade toehold-mediated strand displacement reaction and magnetic beads. Target microRNA-21 acts as an initiator to trigger the cascade toehold-mediated strand displacement reaction and it outputs double-stranded DNA. After magnetic separation, the double-stranded DNA is intercalated by SYBR Green I, resulting in an amplified fluorescent signal. Under the optimal conditions, a wide linear range (0.5-60 nmol/L) and low limits of detection (0.19 nmol/L) are exhibited. What's more, the biosensor shows great specificity and reliability between microRNA-21 and other microRNAs involved in cancer (microRNA-34a, microRNA-155, microRNA-10b, and let-7a). Owing to the properties of fabulous sensitivity, high selectivity, and simplicity of operator, the proposed method paves a promising way for microRNA-21 detection in cancer diagnosis and biological research.

Rapid automatic nucleic acid purification system based on gas-liquid immiscible phase.

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas-liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%-95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.

Development of a separation platform comprising magnetic beads combined with the CRISPR/Cas12a system enabling ultrasensitive and rapid detection of miRNA-21.

A separation platform has been developed mediated by a combination of magnetic beads and the CRISPR/Cas12a system to achieve ultrasensitive and rapid detection of miRNA-21 at a low level. In this system, with the assistance of an auxiliary probe, the target miRNA-21 can be specifically combined with three-stranded probes to initiate the SDR reaction. Abundant aptamer A3 was added to the solution that can activate the CRISPR/Cas12a system and initiate the trans-cleavage reaction to recover the fluorescence signal. Using magnetic beads to mediate the separation considerably greatly improves the signal conversion efficiency and detection sensitivity. At the 492 nm excitation wavelength, and 502-650 nm scan range, through analyzing the fluorescence peak intensity at 520 nm, the biosensor's determination range and limit of detection is 8 fM-250 nM and 2.42 fM, respectively, and the RSD is 19.03-37.80. Compared with other biosensors, the biosensor developed exhibited superior performance and the signal recovered excellently in 1% human serum and the LOD is 12.12 fM. This method provides a novel highly sensitive scheme for detecting miRNA .

Ultra-sensitive detection of tumor necrosis factor alpha based on silver-coated gold core shell and magnetically separated recognition of SERS aptamer sensors.

A highly sensitive tumor necrosis factor α (TNF-α) detection method based on a surface-enhanced Raman scattering (SERS) magnetic patch sensor is reported. Magnetic beads (MNPs) and core shells were used as the capture matrix and signaling probe, respectively. For this purpose, antibodies were immobilized on the surface of magnetic beads, and then Au@4-MBN@Ag core-shell structures coupled with aptamers and TNF-α antigen were added sequentially to form a sandwich immune complex. Quantitative analysis was performed by monitoring changes in the characteristic SERS signal intensity of the Raman reporter molecule 4-MBN. The results showed that the limit of detection (LOD) of the proposed method was 4.37 × 10-15 mg·mL-1 with good linearity (R2 = 0.9918) over the concentration range 10-12 to 10-5 mg·mL-1. Excellent assay accuracy was also demonstrated, with recoveries in the range 102% to 114%. Since all reactions occur in solution and are separated by magnetic adsorption of magnetic beads, this SERS-based immunoassay technique solves the kinetic problems of limited diffusion and difficult separation on solid substrates. The method is therefore expected to be a good clinical tool for the diagnosis of the inflammatory biomarker THF-α and in vivo inflammation screening.