Procalcitonin Detection Using Immunomagnetic Beads-Mediated Surface-Enhanced Raman Spectroscopy.

The early detection of procalcitonin (PCT) is crucial for diagnosing bacterial infections due to its high sensitivity and specificity. While colloidal gold colorimetric and immune-chemiluminescence methods are commonly employed in clinical detection, the former lacks sensitivity, and the latter faces challenges with a brief luminescence process and an elevated background. Here, we introduce a novel approach for the quantitative analysis of PCT using surface-enhanced Raman spectroscopy (SERS), leveraging the enhanced properties of metal nanoparticles. Simultaneously, we employed a magnetic nanoparticle coating and surface biofunctionalization modification to immobilize PCT-trapping antibodies, creating the required immune substrates. The resulting magnetic nanoparticles and antibody complexes, acting as carriers and recognition units, exhibited superparamagnetism and the specific recognition of biomarkers. Then, this complex efficiently underwent magnetic separation with an applied magnetic field, streamlining the cumbersome steps of traditional ELISA and significantly reducing the detection time. In conclusion, the exploration of immunomagnetic bead detection technology based on surface-enhanced Raman spectroscopy holds crucial practical significance for the sensitive detection of PCT.

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.

Aptamer-Based Sensor for Rapid and Sensitive Detection of Ofloxacin in Meat Products.

Ofloxacin (OFL) is widely used in animal husbandry and aquaculture due to its low price and broad spectrum of bacterial inhibition, etc. However, it is difficult to degrade and is retained in animal-derived food products, which are hazardous to human health. In this study, a simple and efficient method was developed for the detection of OFL residues in meat products. OFL coupled with amino magnetic beads by an amination reaction was used as a stationary phase. Aptamer AWO-06, which showed high affinity and specificity for OFL, was screened using the exponential enrichment (SELEX) technique. A fluorescent biosensor was developed by using AWO-06 as a probe and graphene oxide (GO) as a quencher. The OFL detection results could be obtained within 6 min. The linear range was observed in the range of 10-300 nM of the OFL concentration, and the limit of the detection of the sensor was 0.61 nM. Furthermore, the biosensor was stored at room temperature for more than 2 months, and its performance did not change. The developed biosensor in this study is easy to operate and rapid in response, and it is suitable for on-site detection. This study provided a novel method for the detection of OFL residues in meat products.

A rapid immunomagnetic beads-based sELISA method for the detection of bovine αs1-casein based on specific epitopes.

Although αs1-casein poses significant health risks to individuals with milk allergies, the availability of quantification methods for this allergen remains limited. In this study, we developed an immunomagnetic beads-based immunoassay (IMBs-ELISA) for the precise quantitative detection of bovine αs1-CN, specifically targeting epitope AA173-194. No cross-reactivity was observed with the other 7 food allergens including milk allergen. The linear detection range of the established IMBs-ELISA method was 0.125 µg/mL-2.000 µg/mL, with a limit of detection of 0.099 µg/mL. The accuracy of this method was 1.048 %, and the intra-plate and inter-plate precision achieved 4.100 % and 6.777 %, respectively. Notably, the entire IMBs-ELISA process could be completed within 75 min, representing a substantial time-saving advantage over traditional ELISA methods. These results proved the reliability and rapidity of the IMBs-ELISA method for detecting αs1-CN in real food.

Isolation and Purification of Viable PGCs from Mouse Embryos.

In all organisms with sexual reproduction, sperm and oocytes derive from embryonic precursors termed primordial germ cells (PGCs) which pass on genetic information to subsequent generations. Studies aimed to unravel PGC development at molecular level in mammals can be traced at the early 1980s and were hampered by the difficulty in obtaining both sufficient quantities and purity of PGCs. For many laboratories, the isolation and purification methods of PGCs at different stages from embryos are the most shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. In the present chapter, I focus on immunomagnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to 12.5 dpc embryos before their differentiation in oogonia/oocytes in female and prospermatogonia in male.

Magnetic capture device for large volume sample analysis.

Immunomagnetic separation (IMS) techniques employing superparamagnetic particles can successfully isolate various components from mixtures. However, their utility can be limited for large-volume samples, viscous samples, or those containing a high density of particulate matter because of the need to generate high field gradients for particle recovery. Therefore, a new class of immunomagnetic particles was devised utilizing a single, macroscopic Pyrex spinbar conjugated with biorecognition elements to address these limitations. Advantages include an inherent capacity for effective mixing, an almost instantaneous recovery of the spinbar that can be performed without expensive equipment and with no loss of magnetic particles during processing, and reduced transfer of sample matrix. As a result, spinbars can provide an effective means for IMS with large-volume assays composed of complex matrices.

The effect of magnetic bead size on the isolation efficiency of lung cancer cells in a serpentine microchannel with added cavities.

An investigation was conducted to examine the effect of magnetic bead (MB) size on the effectiveness of isolating lung cancer cells using the immunomagnetic separation (IMS) method in a serpentine microchannel with added cavities (SMAC) structure. Carboxylated magnetic beads were specifically conjugated to target cells through a modification procedure using aptamer materials. Cells immobilized with different sizes (in micrometers) of MBs were captured and isolated in the proposed device for comparison and analysis. The study yields significance regarding the clarification of device working principles by using a computational model. Furthermore, an accurate evaluation of the MB size impact on capture efficiency was achieved, including the issue of MB-cell accumulation at the inlet-channel interface, despite it being overlooked in many previous studies. As a result, our findings demonstrated an increasing trend in binding efficiency as the MB size decreased, evidenced by coverages of 50.5%, 60.1%, and 73.4% for sizes of 1.36 µm, 3.00 µm, and 4.50 µm, respectively. Additionally, the overall capture efficiency (without considering the inlet accumulation) was also higher for smaller MBs. However, when accounting for the actual number of cells entering the channel (i.e., the effective capture), larger MBs showed higher capture efficiency. The highest effective capture achieved was 88.4% for the size of 4.50 µm. This research provides an extensive insight into the impact of MB size on the performance of IMS-based devices and holds promise for the efficient separation of circulating cancer cells (CTCs) in practical applications.

Improved biosensing of Legionella by integrating filtration and immunomagnetic separation of the bacteria retained in filters.

A novel approach is presented that combines filtration and the direct immunomagnetic separation of the retained bacteria Legionella in filters, for further electrochemical immunosensing. This strategy allows for the separation and preconcentration of the water-borne pathogen from high-volume samples, up to 1000 mL. The limit of detection of the electrochemical immunosensor resulted in 100 CFU mL-1 and improved up to 0.1 CFU mL-1 when the preconcentration strategy was applied in 1 L of sample (103-fold improvement). Remarkably, the immunosensor achieves the limit of detection in less than 2.5 h and simplified the analytical procedure. This represents the lowest concentration reported to date for electrochemical immunosensing of Legionella cells without the need for pre-enrichment or DNA amplification. Furthermore, the study successfully demonstrates the extraction of bacteria retained on different filtering materials using immunomagnetic separation, highlighting the high efficiency of the magnetic particles to pull out the bacteria directly from solid materials. This promising feature expands the applicability of the method beyond water systems for detecting bacteria retained in air filters of air conditioning units by directly performing the immunomagnetic separation in the filters.

Development of a broad-spectrum one-step immunoassay for detection of glucocorticoids in milk using magnetosome-based immunomagnetic beads.

Immunomagnetic beads provide novel tools for high-throughput immunoassay techniques. In this study, protein G (PG) was immobilized onto bacterial magentic particles (BMPs) using an additional cysteine residue at the C-terminus. A broad-spectrum monoclonal antibody against glucocorticoids (GCs) was attached to BMPs through PG-Fc interaction, generating BMP-PG-mIgG immunomagentic beads. A sensitive one-step immunoassay was developed for GCs based on combination of BMP-PG-mIgG and dexamethasone-horseradish peroxidase tracer (DMS-HRP). The developed assay exhibited half inhibitory concentrations (IC50) for dexamethasone (DMS), betamethasone (BMS), prednisolone (PNS), hydrocortisone (HCS), beclomethasone (BCMS), cortisone (CS), 6-α-methylprednisone (6-α-MPNS), fludrocortisone acetate (HFCS) of 0.98, 1.49, 2.42, 9.29, 1.63, 6.13, 7.3, and 4.89 ng/mL, respectively. The method showed recoveries ranging rates from 86.5 % to 117 % with a coefficient of variation less than 12.3 % in milk sample, which showed a good correlation with LC-MS/MS. Thus, the proposed assay offers a rapid and broad-spectrum screening tool for simultaneous detection of GCs in milk.

Protocol for the isolation and culture of microglia, astrocytes, and neurons from the same mouse brain.

Studying the intrinsic properties of microglia, astrocytes, and neurons is essential to our understanding of brain function. Here, we present a protocol to isolate and culture these neural cells from the same mouse brain. Using immunocapture magnetic beads, we describe steps for dissociating, cleaning, and sequentially separating brains from 9-day-old mice into microglia, astrocytes, and neurons. Following these detailed procedures for seeding and culturing of isolated cells, we can address critical questions related to brain function.

A novel phagomagnetic separation-ATP bioluminescence (PhMS-BL) for rapid and sensitive detection of viable Vibrio parahaemolyticus in aquatic product.

Detection of viable Vibrio parahaemolyticus (V. parahaemolyticus) is a major challenge due to its significant risk to food safety and human health. Herein, we developed a phagomagnetic separation-ATP bioluminescence (PhMS-BL) assay based on phage VPHZ6 for rapid and sensitive detection of viable V. parahaemolyticus. Phage as a recognition element was coupled to magnetic beads to capture and enrich V. parahaemolyticus, shortening detection time and improving method sensitivity. The intracellular ATP released by chemical lysis using CTAB was quantified using firefly fluorescein-adenosine triphosphate bioluminescence system to detect viable bacteria. So, PhMS-BL method was able to detect V. parahaemolyticus in a linear range of 2.3 × 102 to 1.3 × 107 CFU mL-1, with a detection limit of 78 CFU mL-1 within 15 min. It is successfully applied to detect V. parahaemolyticus in spiked lake water, lobster tail meat, and clam meat. The developed detection strategy can rapidly and sensitively detect viable V. parahaemolyticus in food matrixes.

A magnetic microneedle to isolate single immunomagnetically labeled cells.

Immunomagnetic enrichment of cell populations from bodily fluids followed by immunofluorescent labeling is an established sample preparation method often used for the detection and enumeration of rare cells such as circulating tumor cells. For a detailed analysis of the heterogeneous characteristics of these cells, the cells need to be retrieved individually. Although several technologies are available to obtain 100% pure cells either individually or in bulk, these are often expensive, have low specificity, or suffer from high cell losses, either inherent to the technology or caused by sample transfer into special chips. To solve this issue, we introduce the magnetic micro-needle approach, which allows for the isolation of immunomagnetically labeled target cells by the use of a magnetized microneedle directly from glass slides. The magnetic microneedle approach makes use of the already present magnetic labeling used for enrichment, while the glass-slide-based open sample container allows for easy and loss-free sample loading. Additionally, the system facilitates not only the isolation but also the precise placement of cells. As the used parts are low cost, the technology provides researchers with an affordable and efficient method to pick up and isolate, as well as specifically place magnetically labeled cells from enriched fractions, thereby enabling the researchers to isolate or analyze these rare cells in more detail.

Separation of spring viraemia of carp virus from large-volume samples using immunomagnetic beads.

A method for separation of spring viraemia of carp virus (SVCV) from large-volume samples using immunomagnetic beads (IMBs) coated with a polyclonal antibody against SVCV was developed. The optimum amount of IMBs was 2 mg in 100 mL. After IMB treatment, the detection limit of SVCV in reverse transcription quantitative PCR (RT-qPCR) was 103 times the 50% tissue culture infectious dose per mL in 100-mL samples. The concentration of viral RNA extracted from SVCV that had been separated using IMBs was 5.18 × 103-fold higher than that of the unseparated SVCV. When fish samples were tested, the concordance rates of the IMBs/RT-qPCR and RT-qPCR were 100% and 67.5%, respectively.

A Dynamic and Pseudo-Homogeneous MBs-icELISA for the Early Detection of Aflatoxin B1 in Food and Feed.

Aflatoxin B1 (AFB1) is one of the most toxic and harmful fungal toxins to humans and animals, and the fundamental way to prevent its entry into humans is to detect its presence in advance. In this paper, the monoclonal antibody mAbA2-2 was obtained via three-step sample amplification and multi-concentration standard detection using a subcloning method based on the limited dilution method with AFB1 as the target. A dynamic and pseucdo-homogeneous magnetic beads enzyme-linked immunosorbent assay (MBs-icELISA) was established using the prepared antibody as the recognition element and immunomagnetic beads as the antigen carrier. The MBs-icELISA showed good linear correlation in the concentration range of 0.004-10 ng/mL with R2 = 0.99396. The limit of detection (LOD) of the MBs-icELISA for AFB1 was 0.0013 ng/mL. This new ELISA strategy significantly shortened AFB1 detection time through improved sensitivity compared to the conventional ELISA method.

Comparison of four commercial immunomagnetic separation kits for the detection of Cryptosporidium.

Cryptosporidium spp. are protozoan parasites of significant health importance found in environmental waters globally. Four commercially available Cryptosporidium-specific immunomagnetic separation (IMS) kits used in various water sample matrices were analysed and compared. Beads were characterised by flow cytometry and tested for the recovery efficiencies for oocysts spiked into different matrices: river water sediment, clay sample, and filter backwash sample. Results showed that Dynabeads™ Cryptosporidium and Waterborne Crypto-Grab™ kits contained immunoglobulin IgM antibody-coated beads. In contrast, the BioPoint CryptoBead and the TCS Isolate kits contained immunoglobulin IgG antibody-coated beads. BioPoint CryptoBead was significantly coated with more antibodies and were able to capture oocysts more rapidly compared to the other beads. Recovery efficiencies of Dynabeads™, TCS Isolate® beads, and BioPoint CryptoBead ranged from 55 to 93% when tested against different sample matrices, with BioPoint CryptoBead resulting in the highest at 93% in reagent-grade water and Dynabeads™ at 55%, the lowest against clay samples. The Waterborne beads did not perform well on any samples, with recovery efficiencies ranging from 0 to 8%. Fluorescence microscopy analyses showed that both the IMS method and the sample matrix processed affect the quality of the membranes, with the cleanest samples for microscopy examination observed from BioPoint CryptoBead.

Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.

Separation mixed semen of two individuals using magnetic beads coupled ABH blood group antibody.

In sexual assault cases, one of the most common samples collected is a mixed semen stain, which is often found on the vagina, female underwear, or bed sheets. However, it is usually difficult to identify the perpetrator based on this sample alone. One technique that has been developed to address this issue is magnetic bead-based separation. This method involves using modified magnetic microspheres to capture and enrich specific target cells, in this case, sperm cells. In this study, we utilized magnetic beads coupled with ABH blood group antibody to isolate sperm cells from an individual of a single ABO blood type. Subsequently, polymerase chain reaction amplification and capillary electrophoresis were employed to perform the genotyping the short tandem repeat (STR) loci. This approach allows for the identification of different individuals in a mixed seminal stain sample from two individuals, by first separating sperm cells based on ABH antigen differences and subsequently utilizing autosomal STR typing on the enriched single blood group cells.

Subtype specific virus enrichment with immunomagnetic separation method followed by NGS unravels the mixture of H5 and H9 avian influenza virus.

Wild bird avian influenza type A virus (AIV) surveillance is important for the early detection of highly pathogenic AIVs and for providing early warnings to the poultry industry and veterinary services to implement more effective control measures against these viruses. Some field samples are often found to contain more than two kinds of AIV. Correct determination of the HA/NA subtype and complete nucleotide sequences of the component viruses in those samples are often critical for timely and accurate understanding of the field situation, but it is not easy to define the genomic structure of the constituent viruses unambiguously because AIV has eight segmented genomes. In this study, with immunomagnetic beads incorporating polyclonal antibodies of chicken for subtype-specific viral enrichment, we could selectively decrease the density of one of the two constituent viruses in a sample of different subtypes, H5 and H9, artificially generated; this was represented in the changes of Ct values with subtype specific real-time RT-PCR. Following this, with NGS, we could recover nearly complete genomic sequences and arrange the consensus sequences of gene segments of the constituent viruses confidently with the quantitative variable like genome coverage, linked along the gene segments and associated with the number of viral copies in a sample.

Boosting SARS-CoV-2 Enrichment with Ultrasmall Immunomagnetic Beads Featuring Superior Magnetic Moment.

The isolation and enrichment efficiency of SARS-CoV-2 virus in complex biological environments is often relatively low, presenting challenges in direct detection and an increased risk of false negatives, particularly during the early stages of infection. To address this issue, we have developed a novel approach using ultrasmall magnetosome-like nanoparticles (≤10 nm) synthesized via biomimetic mineralization of the Mms6 protein derived from magnetotactic bacteria. These nanoparticles are surface-functionalized with hydrophilic carboxylated polyethylene glycol (mPEG2000-COOH) to enhance water solubility and monodispersity. Subsequently, they are coupled with antibodies targeting the receptor-binding domain (RBD) of the virus. The resulting magnetosome-like immunomagnetic beads (Mal-IMBs) exhibit high magnetic responsiveness comparable to commercial magnetic beads, with a saturation magnetization of 90.6 emu/g. Moreover, their smaller particle size provides a significant advantage by offering a higher specific surface area, allowing for a greater number of RBD single-chain fragment variable (RBD-scFv) antibodies to be coupled, thereby enhancing immune capture ability and efficiency. To validate the practicality of Mal-IMBs, we evaluated their performance in recognizing the RBD antigens, achieving a maximum capture ability of 83 µg/mg per unit mass. Furthermore, we demonstrated the binding capability of Mal-IMBs to SARS-CoV-2 pseudovirus using fluorescence microscopy. The Mal-IMBs effectively enriched the pseudovirus at a low copy concentration of 70 copies/mL. Overall, the small Mal-IMB exhibited excellent magnetic responsiveness and binding efficiency. By employing a multisite virus binding mechanism, it significantly improves the enrichment and separation of SARS-CoV-2 in complex environments, facilitating rapid detection of COVID-19 and contributing to effective measures against its spread.

Rapid detection of multiple antibiotics in chicken samples via a fluorescence nanobiosensor coupled with a homemade fluorescence analyzer.

Antibiotic residues in foods pose a serious threat to human health. However, routine analysis techniques require bulky laboratory instruments and skilled personnel or give single-channel analysis results, exhibiting low practicality. Here, we explored a rapid and easy-to-use detection system combining a fluorescence nanobiosensor with a homemade fluorescence analyzer for the simultaneous identification and quantification of multiple antibiotics. The nanobiosensor assay worked based on the targeted antibiotics competing with signal labels of antigen-quantum dots (IQDs) to bind with recognition elements of antibody-magnetic beads (IMBs). The fluorescence signals of IMB-unbound IQDs in a magnetically separated supernatant, related to antibiotic concentration, were automatically collected and processed by our self-designed and homemade fluorescence analyzer which integrated mechanical control hardware (consisting of a mechanical arm, a ten-channel rotary bench, and an optical detection unit) and user control software (installed on a built-in laptop). The fluorescence analyzer enabled the analysis of 10 samples within 5 min in one round and permitted the real-time uploading of sample data to the cloud. By employing three QDs with emission wavelengths of 525 nm, 575 nm, and 625 nm, this multiplex fluorescence biosensing system demonstrated great sensitivity and accuracy for simultaneously analyzing enrofloxacin, tilmicosin, and florfenicol in chicken samples with detection limits of 0.34 µg kg-1, 0.7 µg kg-1, and 0.16 µg kg-1, respectively. Moreover, the biosensing platform performed well in a wealth of chicken samples covering various breeds from three Chinese cities. This study identifies a generic and user-friendly multiplex biosensor platform with significant potential for use in food safety and regulation.