Preparation of immunomagnetic iron-dextran nanopartides and application in rapid isolation of E.coli O157:H7 from foods

AIM: To prepare a kind of magnetic iron-dextran nanopartides that was coated with anti-E.coli O157:H7 IgG, analyze its application conditions, and try to use it to isolate E.coli O157:H7 from foods. METHODS: Magnetic iron-dextran nanopartides were prepared by the reaction of a mixture of ferric and ferrous ions with dextran polymers under alkaline conditions. The particles were coated with antiserum against E.coli O157: H7 by the periodate oxidation-borohydride reduction procedure. The oxidation time, amount of antibody coating the particles, amount of nanoparticles, incubation time and isolation time were varied to determine their effects on recovery of the organisms. Finally, the optimum conditions for isolating E.coli O157:H7 from food samples were established. RESULTS: E.coli O157:H7 can be isolated from samples within 15 min with the sensitivity of 101 CFU/mL or even less. In the presence of 108 CFU/mL of other organisms, the sensitivity is 101-102 CFU/mL. Nonspecific binding of other bacteria to the particles was not observed. Two and a half hours of enrichment is enough for the particles to detect the target from the food samples inoculated with 1 CFU/g. CONCLUSION: Isolation of target bacteria by immuno magnetic nanoparticles is an efficient method with high sensitivity and specificity. The technique is so simple that it can be operated in lab and field even by untrained personnel.

Immunomagnetic removal of cryo-damaged human spermatozoa

Aim: To estimate the dissipation of mitochondrial transmembrane potential (mTMR,Δψ_m) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding. Methods: The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB). Results: The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1% ± 7.2 % to 53.5 % ± 13.1% and increased the spermatozoa with activated pancaspases (aCP) from 21.8 % ± 2.6 % to 47.7 % ± 5.8 % (n = 10; mean ± SEM; P < 0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n = 22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 ± 3.9 (P < 0.01) and also those with aCP to 9.3 % ± 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P < 0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions. Conclusion: The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

Preparation of Immunomagnetic Beads Enrichment Kit for Detection of Aflatoxin B1

Immunomagnetic beads enrichment kit for detection of aflatoxin B1(AFB1) was prepared through reaction of AFB1 and p-phenylenediamine. The catches of AFB1 by the kit were 25 ng/mg. Furthermore, AFB1 was conducted specific reaction with competitive drugs with similar structure or function to AFB1, including aflatoxin M1, T-2 toxin, ochratoxin A, zearalenone and patulin, and no cross reaction was observed.

Development of a Kind of Immunomagnetic Bead Kit to Separate and Enrich Salbutamol

An immunomagnetic bead kit to separate and enrich salbutamol was prepared through the reaction between salbutamol and 4-aminobenzoic acid.The kit had a catch of 20 ng/ml to salbutamol in samples,and showed no cross reaction with competitive drugs with structure and function similar to salbutamol: clenbuterol,ractopamine,phenylethanolamine A, bromchlorbuterol, brombuterol, terbutaline, hydroxymethyl salbutamol,cimaterol,tulobuterol,mapenterol,cimbuterol,clenpenterol,zilpaterol,penbutolol,clenproperol,mabuterol and clorprenaline.

Immunomagnetic assay combined with CdSe/ZnS amplification of chemiluminescence for the detection of abscisic acid

Abscisic acid (ABA) is an important plant hormone. It plays a key role in regulating plant responses to abiotic stress and in controlling seed germination, growth, and stomatal aperture. A rapid, sensitive analytical method for the ABA detection is urgently required for further investigation of ABA signaling. In this work, an immunomagnetic assay combined with CdSe/ZnS amplification of chemiluminescence has been developed for the detection of ABA. The result could be read out in 30 min at least, with the simplified procedure of immunomagnetic assay. Under the optimized condition, a linear range from 1 pM to 10 nM was obtained. An unexpected result induced by the dose hook effect was discussed. This method provided the high selectivity for ABA over other components that might be contained in real samples.

On-chip immunomagnetic bead swarm based on magnetic actuation and mechanical vibration for biological detection

Immunomagnetic bead(IMB)-based detection has great potential for biomedical applications.Passive and active strategies,including microfluidics and magnetic actuation methods,have been developed to mix IMBs and analytes efficiently.However,cost-effective on-site detection using a simple microfluidic chip is challenging,and miniaturization of the magnetic driving device is imperative for portability.In this study,we propose a novel mixing method for an on-chip IMB swarm via magnetic actuation and mechanical vibration.A microfluidic chip system coupled with double spiral magnetic coils and a vibration motor was fabricated.The aggregation behavior of IMBs under magnetic fields and the diffusion behavior of the IMB swarm under mechanical vibration were analyzed in detail.Based on the synergetic effects of magnetic actuation and mechanical vibration,we achieved the highly efficient capturing of Vibrio parahaemolyticus DNA and goat anti-human immunoglobulin G by mixing the IMB swarm with the microfluidic chip.In this case,the antigen detection rate could reach~94.4%.Given its fascinating features,such IMB-microfluidic detection demonstrates significant potential for biomedical applications.

Isolation,cultivation and identification of brain glioma stem cells by magnetic bead sorting

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

Study on Shigella Detection by ATP Bioluminescence Magnetic Enzyme Immunoassay

[Objective] The research aimed to establish a rapid detection method for Shigella. [Method] Combining immunomagnetic separation technology with ATP bioluminescence technology, a new kind of fast and accurate ATP bioluminescence magnetic enzyme immunoassay technique for Shigella was established. [Result] Using ATP bioluminescence magnetic enzyme immunoassay technique to detect standard solution for Shigella （ATCC 25931 ）, result showed that correlation coefficient between relative light intensity detected by instrument and bacteria concentration detec- ted by culture counting method was 0.981 1. Moreover, relation curve between relative light intensity and Shigella concentration was drawn. [ Conclusion] The method had a high detection speed and accuracy, and could be used for the rapid detection of pathogen in food and environment.

Separation of CD34 Positive Cells and Determination of Surface Homing Antigen

To study the differences in homing potential between bone marrow cells and umbilical blood cells, CD34 positive cells were obtained from bone marrow (BM) and umbilical blood (UB) by the direct cell separation with domestic immunomagnetic beads. The expression of the two adhesion molecules CD11a/CD18 and CD44 were examined. After separation, CD34 positive cells accounted for 51 %-82 % of the harvested cells and dye-resistance rate was 82 %-88 %. The expression of CD11a/CD18 and CD44 on the surfaces of UB cells was 49. 6 % 1 10. 2 % and 37. 7 % ± 10. 3 % respectively. On BM cells they were 50. 2 % ± 6. 2 % and 34 % ± 13. 3 % respectively. There were no significant differences in the expression of these two molecules. It was concluded .that the cell separation method with domestic immunomagnetic beads was effective and the stem cells from UB could serve as an alternative source for transplantation.

Development of Annexin V Technology for Removal of Dead Spermatozoa from Bovine Spermatozoa and Its Application in Frozen Sexed Semen Production

[Objectives]This study was conducted to develop a molecular marker immunomagnetic bead sorting technology method that can specifically identify dead spermatozoa.[Methods]This study first confirmed the specific binding of Annexin V to dead bovine spermatozoa,and tried to remove dead spermatozoa in semen combining with the immunomagnetic bead technology,so as to improve the separation efficiency of target spermatozoa in the process of sex-controlled semen preparation on a flow cytometer.[Results]The spermatozoon motility,membrane integrity and mitochondrial activity after sorting and the rate of dead spermatozoa during the on-machine X/Y separation were all improved to different degrees(P<0.05),indicating that the technical process design could effectively remove some dead spermatozoa,and there was no significant effect on frozen sexed semen prepared from the separated X or Y spermatozoa(P>0.05),indicating that the technical process did not cause additional damage to the spermatozoa.[Conclusions]Combining the specificity of Annexin V with the immunomagnetic bead method could effectively remove dead spermatozoa from bovine spermatozoa,and significantly reduce the rate of dead spermatozoa in bovine permatozoa during sex-controlled separation(P<0.05).The method developed can effectively improve the production efficiency of frozen sexed semen of dairy cows,reduce the production cost,and promote the industrial application of the product.

Culture Age on Evaluation of Electrically Active Magnetic Nanoparticles as Accurate and Efficient Microbial Extraction Tools

A potential confounding factor in the development and evaluation of biosensors is the diverse nature of the disciplines involved. Biosensor technology involves electrochemistry, microbiology, chemical synthesis, and engineering, among many other disciplines. Biological systems, due to non-homogeneous distribution, are already imprecise compared with other systems, especially food based systems. Inadequate knowledge of the techniques to moderate this leads to ineffective evaluation strategies and potentially halting the pursuit of excellent technology that was merely poorly evaluated. This research was undertaken to evaluate the effect culture age had on the capture efficiency of the electrically active magnetic nanoparticles (EAMNP) using culture as the evaluation tool. The age of culture used for immunomagnetic separation (IMS) over all the experiments was 6 to 18 hours. Ideal culture age range for evaluating biosensors is 4 to 10 hours according to the growth curve for E. coli O157: H7 in trypticase soy broth. This is supported by the statistically significant difference among organisms in groups from 3 to 10 hours old compared with those grouped from 11 to 18 and >19 hours old (α = 0.05, p = 0.001 and p = 0.014 respectively). The two older categories were not different from each other. The capture efficiency in all biosensor analysis will vary less than when culture of only viable cells is the diagnostic tool. This allows a true evaluation of the consistency and accuracy of the method, less hindered by the variation in the ability to culture the organism.

Platinum Nanoparticle-based Collision Electrochemistry for Rapid Detection of Breast Cancer MCF-7 Cells

Cancer metastasis is the leading cause of death in cancer patients worldwide and one of the major challenges in treating cancer.Circulating tumor cells(CTCs)play a pivotal role in cancer metastasis.However,the content of CTCs in peripheral blood is minimal,so the detection of CTCs in real samples is extremely challenging.Therefore,efficient enrichment and early detection of CTCs are essential to achieve timely diagnosis of diseases.In this work,we constructed an innovative and sensitive single-nanoparticle collision electrochemistry(SNCE)biosensor for the detection of MCF-7 cells(human breast cancer cells)by immunomagnetic separation technique and liposome signal amplification strategy.Liposomes embedded with platinum nanoparticles(Pt NPs)were used as signal probes,and homemade gold ultramicroelectrodes(Au UME)were used as the working electrodes.The effective collision between Pt NPs and UME would produce distinguishable step-type current.MCF-7 cells were accurately quantified according to the relationship between cell concentration and collision frequency(the number of step-type currents generated per unit time),realizing highly sensitive and specific detection of MCF-7 cells.The SNCE biosensor has a linear range of 10 cells·mL^(-1)to 10^(5) cells·mL^(-1)with a detection limit as low as 5 cells·mL^(-1).In addition,the successful detection of MCF-7 cells in complex samples showed that the SNCE biosensors have great potential for patient sample detection.

Improvement of detection method of Cryptosporidium and Giardia in reclaimed water

Cryptosporidium and Giardia are two typical species of pathogenic protozoans that seriously endanger water quality.Previous works indicated that detection of Cryptosporidium and Giardia with modified United States Environmental Protection Agency(USEPA)method-1623 using a membrane filtration-elution for sample concentration attained better recovery and lower cost compared to the USEPA method-1623.Several improvements of membrane filtration-elution step as well as immunomagnetic separation(IMS)steps were investigated and an optimized method for detection of Cryptosporidium and Giardia in wastewater reclamation system was recommended in this paper.The experimental results show that an overnight soak of the membrane after scraping and vortex agitation before elution could enhance and stabilize the recovery.Increasing turbidity to 4 NTU by adding kaolin clay before filtration could effectively improve the recovery of low-turbidity water.Washing the concentrate after centrifugation and twice acid dissociation both reduced the impact of water quality to protozoan recovery.Protozoans in different water samples were determined by this optimized method,and the recovery of Cryptosporidium and Giardia were above 70% and 80%respectively,much higher than the acceptance of method-1623.

Novel aerosol detection platform for SARS–CoV–2:Based on specific magnetic nanoparticles adsorption sampling and digital droplet PCR detection

The SARS–CoV–2 virus is released from an infectious source(such as a sick person)and adsorbed on aerosols,which can form pathogenic microorganism aerosols,which can affect human health through airborne transmission.Efficient sampling and accurate detection of microorganisms in aerosols are the premise and basis for studying their properties and evaluating their hazard.In this study,we built a set of sub-micron aerosol detection platform,and carried out a simulation experiment on the SARS–CoV–2 aerosol in the air by wet-wall cyclone combined with immunomagnetic nanoparticle adsorption sampling and ddPCR.The feasibility of the system in aerosol detection was verified,and the influencing factors in the detection process were experimentally tested.As a result,the sampling efficiency was 29.77%,and extraction efficiency was 98.57%.The minimum detection limit per unit volume of aerosols was 250 copies(102copies/m L,concentration factor 2.5).

Nominal effective immunoreaction volume of magnetic beads at single bead level

Immunomagnetic bead（IMB）-based enzyme-linked immunosorbent assay（ELISA） has been the tool frequently used for protein detection in research and clinical laboratories.For most ELISA reactions the recommended dosage of IMBs is usually according to their weight（mg） or mass fraction（w/v） instead of the bead number.Consequently, the processes occurring in the immediate vicinity of the IMBs have always been ignored by researchers and they cannot be revealed in detail during the ELISA reaction.In this paper, we established the relationship between number of IMBs and colorimetric results, and further proposed a new concept of ＂nominal effective immunoreaction volume（NEIV）＂ to characterize a single IMB during ELISA reaction.Results showed that the NEIV of a single IMB has a constant value, which is unrelated to the amount of beads and the concentration of antigen.Optimal results of the colorimetric ELISA are achieved when the incubation volume meets each IMB＇s NEIV and is no longer enhanced by increasing the incubation volume.Thus, the reliable and relatively precise number of IMBs for ELISA detection during practical application could be determined.Most importantly, a study using IMB＇s NEIV would lay the foundation for a kinetics analysis of IMBs and antigens for future study.

Fe_(3)O_(4)@SiO_(2)-Protein A-oHSV/CD63 Ab for Capturing Virus and Exosomes

Protein A modified magnetic spheres(Fe_(3)O_(4)@SiO_(2)-Protein A)with 22.7 emu/g saturation magnetization were prepared and characterized.These spheres had a diameter of 10 nm and was stable up to 229.2℃,and can bond 19.694 mg/g of Protein A.The Fe_(3)O_(4)@SiO_(2)-Protein A was firstly combined with oHSV-BJ-2-A antibody to capture oHSV from disease venom,and the amount of binding virus was 1.92×10^(7)to 3.44×10^(7)PFU/g.Fe_(3)O_(4)@SiO_(2)-Protein A was modified with CD63 antibody,which was further fixed using dissuccinimide octylate(DSS)as a crosslinking agent.The prepared Fe_(3)O_(4)@SiO_(2)-Protein A-CD63 Ab was useful in isolating and enriching exosomes from cell supernatant.Compared with commercial kits,Fe_(3)O_(4)@SiO_(2)-Protein A-CD63 Ab demonstrated a better purification effect,which was successfully monitored using capillary electrophoresis.The total content of exosomes protein extracted using Fe_(3)O_(4)@SiO_(2)-Protein A-CD63 was 3.4 mg/g,which was obviously higher than that reported in some studies.In addition,Fe_(3)O_(4)@SiO_(2)-Protein A-CD63 Ab also showed its repeatability through bonding and elution of 5 cycles,which was effective in cost saving.These indicated Fe_(3)O_(4)@SiO_(2)-Protein A-CD63 Ab had the potential for large-scale purification of exosomes in practical applications.