Functional Nanoparticles with Magnetic 3D Covalent Organic Framework for the Specific Recognition and Separation of Bovine Serum Albumin.

Glutathione functionalized magnetic 3D covalent organic frameworks combined with molecularly imprinted polymer (magnetic 3D COF-GSH MIPs) were developed for the selective recognition and separation of bovine serum albumin (BSA). Ultrasonication was used to prepare magnetic 3D COFs with high porosity (~1 nm) and a large surface area (373 m2 g-1). The magnetic 3D COF-GSH MIP nanoparticles had an imprinting factor of 4.79, absorption capacity of 429 mg g-1, magnetic susceptibility of 32 emu g-1, and five adsorption-desorption cycles of stability. The proposed method has the advantages of a shorter equilibrium absorption time (1.5 h), higher magnetic susceptibility (32 emu g-1), and larger imprinting factor (4.79) compared with those reported from other studies. The magnetic 3D COF-GSH MIPs used with BSA had selectivity factors of 3.68, 2.76, and 3.30 for lysozyme, ovalbumin, and cytochrome C, respectively. The successful recognition and separation of BSA in a real sample analysis verified the capability of the magnetic 3D COF-GSH MIP nanoparticles.

Rapid Preparation of Fluorescent Carbon Dots from Pine Needles for Chemical Analysis.

Fluorescent carbon dots with blue, green, and red emissions were rapidly prepared from modified pine needles through microwave irradiation in a one-pot reaction. The fluorescence intensity and emission versatility for a carbon source were experimentally optimized. The reaction times were under 10 min and the reaction temperatures were lower than 220 °C. Potential applications of magnetic fluorescence-linked immunoassays of carcinoembryonic antigen (CEA) and tumor necrosis factor-alpha (TNF-α) were presented. The detection limits for CEA and TNF-α (3.1 and 2.8 pg mL-1, respectively) are lower than those presented in other reports, whereas the linear ranges for CEA and TNF-α (9 pg mL-1 to 18 ng mL-1 and 8.5 pg mL-1 to 17 ng mL-1, respectively) are wider than those presented in other reports. Magnetic immunoassays with fluorescent CDs prepared from pine needles can enable rapid, sensitive, and selective detections for biochemical analysis.

Microwave Synthesis of Gold Nanoclusters with Garlic Extract Modifications for the Simple and Sensitive Detection of Lead Ions.

Novel bovine serum albumin (BSA)-gold nanoclusters with garlic extract modifications (mw_G-BSA-AuNCs) were prepared through microwave-assisted rapid synthesis. The modified nanoclusters were characterized and used for the simple and sensitive detection of Pb2+ ions. Both turn-on and turn-off methods were used and compared for Pb2+ ion detection. For Pb2+ ions, the preparation time for the modified nanoclusters was 10 min, and the detection time for the nanoclusters was 6 min. The modified nanoclusters were stable, and their fluorescence intensities changed by less than 10% in 60 days. The detection limit and linear range for the "off-on" method of mw_G-BSA-AuNCs for Pb2+ ion detection were 0.28 and 1-20 nM, respectively. The recoveries of the mw_G-BSA-AuNCs probe used for the detection of the Pb(II) ion in tap water ranged from 93.8% to 102.2%, with an average of 97.1%. The "off-on" method of mw_G-BSA-AuNCs can provide a lower detection limit, higher selectivity, and better recovery than the commonly used "turn-off" methods of mw_BSA-AuNCs for Pb2+ ion detection. The proposed method is superior to other methods proposed from 2018 to 2019 because it can provide a shorter preparation time and a lower detection limit with good selectivity. The microwave-assisted novel compound, mw_G-BSA-AuNCs, can be rapidly synthesized in a green manner and can provide a low detection limit, good selectivity, and a simple and fast reaction for Pb2+ ion detection.

A magneto-microfluidic platform for fluorescence immunosensing using quantum dot nanoparticles.

This study reports the online fluorescent detection of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) biomarker proteins in microfluidic channels using functional nanoparticles. Functional magnetic nanoparticles labeled with two antibodies were predeposited on separated microfluidic channels. Antigens were passed through each microfluidic channel to react with the respective antibodies. Two types of fluorescent nanoparticles labeled with antibodies were then used to detect and confirm antigens in the immunocomplex. Results indicate that online fluorescent detection of proteins can provide advantages for real-time monitoring and diagnostic applications. The running time was less than 20 min for each trial. The detection limits of CEA and AFP were found to be 0.6 and 0.2 pg ml-1. These detection limits are lower than those of ELISA. The linear ranges of CEA and AFP detection were from 1.8 pg ml-1 to 1.8 ng ml-1 and from 0.68 pg ml-1 to 0.68 ng ml-1 for two deposition zones in a magnetic sandwich immunoassay. The linear ranges of this method are wider than those of ELISA and those of most other methods. The measurements of CEA and AFP in serum samples from this method differed from ELISA results by 11% and 9.4%, respectively. The detection limit of online detection has achieved the same range as those of previous offline detection. This method has a good potential for automation and multichannel analysis to increase the throughput with some modifications in the future. The proposed method can provide simple, fast, and sensitive online detection for biomarkers.

Magnetofluorescent nanocomposites and quantum dots used for optimal application in magnetic fluorescence-linked immunoassay.

Magnetofluorescent nanocomposites with optimal magnetic and fluorescent properties were prepared and characterized by combining magnetic nanoparticles (iron oxide@polymethyl methacrylate) with fluorescent nanoparticles (rhodamine 6G@mSiO2). Experimental parameters were optimized to produce nanocomposites with high magnetic susceptibility and fluorescence intensity. The detection of a model biomarker (alpha-fetoprotein) was used to demonstrate the feasibility of applying the magnetofluorescent nanocomposites combined with quantum dots and using magnetic fluorescence-linked immunoassay. The magnetofluorescent nanocomposites enable efficient mixing, fast re-concentration, and nanoparticle quantization for optimal reactions. Biofunctional quantum dots were used to confirm the alpha-fetoprotein (AFP) content in sandwich immunoassay after mixing and washing. The analysis time was only one third that required in ELISA. The detection limit was 0.2 pg mL-1, and the linear range was 0.68 pg mL-1-6.8 ng mL-1. This detection limit is lower, and the linear range is wider than those of ELISA and other methods. The measurements made using the proposed method differed by less than 13% from those obtained using ELISA for four AFP concentrations (0.03, 0.15, 0.75, and 3.75 ng mL-1). The proposed method has a considerable potential for biomarker detection in various analytical and biomedical applications. Graphical abstract Magnetofluorescent nanocomposites combined with fluorescent quantum dots were used in magnetic fluorescence-linked immunoassay.

Multifunctional nanoparticles for protein detections in thin channels.

This paper presents a method for simultaneous detection of two proteins by using multifunctional nanoparticles with a magnetic immunoassay in thin channels. Biofunctional magnetic graphene quantum dots (GQDs) combined with two biofunctional quantum dots (QDs) were used for simultaneously detecting two proteins. Magnetic GQDs enabled selective and quantitative nanoparticle deposition with blue emission. Biofunctional QDs confirmed the two protein detections with orange and green emissions. We used two model biomarkers [alpha-fetoprotein (AFP) and cancer antigen 125 (CA125)] to demonstrate the feasibility of the proposed method. The detection limits (0.06pg/mL AFP and 0.001U/mL CA125) and linear ranges (0.2pg/mL-0.68ng/mL AFP and 0.003-25U/mL CA125) of this method are the same as those of single protein detection within experimental errors. These detection limits are substantially lower and the linear ranges are considerably wider than those of enzyme-linked immunosorbent assay (ELISA) and other immunoassay methods. The differences between the proposed method and an ELISA method in AFP and CA125 measurements of serum samples were less than 12%. The proposed method demonstrates favorable detection of biomarkers with advantages of speed, sensitivity, selectivity, and throughput.

Estimation of Molecular Interaction Force Using Atomic Force Microscopy for Bioapplication.

We report a method that involves using atomic force microscopy to estimate molecular interaction forces for bioapplications. Experimental parameters, comprising the labeling concentrations of tips and substrates and the loading rates of tips, were optimized for estimating molecular interaction forces for three pairs of model molecules (IgG/anti-IgG, BSA/anti-BSA, streptavidin/biotin). The estimated molecular interaction forces of IgG/anti-IgG, BSA/anti-BSA, and streptavidin/biotin were 121 ± 3, 185 ± 4, and 241 ± 4 pN, respectively. The measured values were consistent and within the range of values reported in the literature. Estimation of molecular interaction forces in force-distance curves for bioapplication is still challenging. There are many potential bioapplications with further investigations. Providing additional screening reference for microsensing applications is one example. This method demonstrates favorable potential for effectively estimating molecular interaction forces for various applications of protein-ligand, antibody-antigen, ligand-receptor complexes, and other bioreactions. This method is also useful for studies of the structures and properties of molecular, cellular, and bacterial surfaces.

Detection of c-reactive protein based on a magnetic immunoassay by using functional magnetic and fluorescent nanoparticles in microplates.

We report the preparation and application of biofunctional nanoparticles to detect C-reactive protein (CRP) in magnetic microplates. A CRP model biomarker was used to test the proposed detection method. Biofunctional magnetic nanoparticles, CRP, and biofunctional fluorescent nanoparticles were used in a sandwich nanoparticle immunoassay. The CRP concentrations in the samples were deduced from the reference plot, using the fluorescence intensity of the sandwich nanoparticle immunoassay. When biofunctional nanoparticles were used to detect CRP, the detection limit was 1.0 ng ml(-1) and the linear range was between 1.18 ng ml(-1) and 11.8 µg ml(-1). The results revealed that the method involving biofunctional nanoparticles exhibited a lower detection limit and a wider linear range than those of the enzyme-linked immunosorbent assay (ELISA) and most other methods. For CRP measurements of serum samples, the differences between this method and ELISA in CRP measurements of serum samples were less than 13%. The proposed method can reduce the analysis time to one-third that of ELISA. This method demonstrates the potential to replace ELISA for rapidly detecting biomarkers with a low detection limit and a wide dynamic range.

Electrochemical sensing of hepatocyte viability.

We investigated the use of amperometric and chronoamperometric methods with a double mediator system and screen-printed electrodes (SPEs) for the electrochemical sensing of hepatocyte viability. Cell counts were determined based on measuring cellular respiration via interaction of electroactive redox mediators. The oxidation currents of chronoamperometric measurement were proportional to the concentrations of ferrocyanide which was produced via interaction of cellular respiration, succinate and ferricyanide. The integrated oxidation charges increased linearly with the density of the cultured primary rat hepatocytes over a range of 1 × 10(5) to 5 × 10(5) cells per well (slope = 1.98 (±0.08) µC per 10(5) cells; R(2) = 0.9969), and the detection limit was 7600 (±300) cells per well based on S/N = 3. Each density of cells was cultured in triple replicates and individual cell samples were evaluated. The results of the cytotoxic effect of the chronoamperometric method are comparable to those of the tetrazolium-based colorimetric assay. The chronoamperometric method with ferricyanide and succinate mediators is an efficient, alternative method for assessing the viability of primary hepatocytes which can be completed in 20 min. Succinate did not provide an efficient electron shuttle between cytosolic respiratory redox activity of cancer cells and extracellular ferricyanide, an effect that may be useful for distinguishing hepatocarcinoma cells from healthy hepatocytes.

Effects of particle characteristics on magnetic immunoassay in a thin channel.

The effects of size and porosity of particles on magnetic immunoassay in a thin channel were studied. Experimental parameters were investigated and compared using a model immunoassay complex of carcinoembryonic antigen (CEA)/anti-CEA. The rate constant for the affinity reaction between functional particles increased as the size of magnetic nanoparticles (800-80 nm) decreased. The affinity reaction between functional particles had no significant effect on the sizes of microparticles (1.0-4.4 µm) at commonly used thin channel flow-rates of 0.001-0.025 ml/min. Competitive and sandwich reactions of CEA/anti-CEA were studied for CEA detection. Microparticles of different porosities produced similar linear ranges of detection and limits of detection. The limits of detection for CEA were 0.29 pg/ml and 0.21 pg/ml for competitive and sandwich reactions, respectively. The linear ranges of detection were from 0.49 pg/ml to 4.9 ng/ml for both competitive and sandwich reactions. The detection limits were lower, and the linear ranges were wider than those of literature. There was a 9% difference in CEA detection measurements between competitive and sandwich magnetic immunoassay. The measurements of two magnetic immunoassays differed by less than 13% from the ELISA reference measurements. The running time was less than 30 min. Magnetic immunoassay in a thin channel has great potential for biochemical analysis and immunoassay-related applications.

Determination of hepatitis B surface antigen using magnetic immunoassays in a thin channel.

We report novel methods for detection of hepatitis B surface antigen (HBsAg) based on competitive and sandwiched magnetic immunoassays using functional magnetic nanoparticles in a thin channel. Magnetic nanoparticles labeled with hepatitis B antibody are flowed through a thin channel to form a predeposition layer for capturing HBsAg. Competitive and sandwiched magnetic immunoassays were studied and detection limit, linear range, and sample selectivity were compared. The detection limits of competitive and sandwiched magnetic immunoassays were found to be 0.26 and 0.25 pg/ml, respectively. The linear range of HBsAg concentration was 0.26 pg/ml-2.6 ng/ml for competitive magnetic immunoassay and was 0.89 pg/ml-8.9 ng/ml for sandwiched magnetic immunoassay. The advantages of these methods over ELISA and other methods for HBsAg detection are lower detection limits and wider linear ranges. The running time was less than 30 min. Competitive magnetic immunoassay was faster than sandwiched magnetic immunoassay for detection of HBsAg. The measurements of HBsAg in serum samples from these methods differed by about 10% from those of ELISA. These methods can provide simple, fast, and sensitive detections of biomarkers and other immunoassay-related samples.

Competitive magnetic immunoassay for protein detection in thin channels.

Functional magnetic nanoparticles are prepared and characterized for protein detection in a magnetic separation channel. This detection method is based on a competitive immunoassay of magnetic separation in thin channels using functional magnetic nanoparticles. We used protein A-IgG complex to demonstrate the feasibility. Free IgG and fixed number of IgG-labeled microparticles were used to compete for limited sites of protein A on the magnetic nanoparticles. Several experimental parameters were investigated for protein detection. The deposited percentages of IgG-labeled microparticles at various concentrations of free IgG were determined and used as a reference plot. The IgG concentration in a sample was deduced and determined based on the reference plot using the deposited percentage of IgG-labeled microparticles from the sample. The linear range of IgG detection was from 5.0 x 10(-8) to 1.0 x 10(-11) M. The detection limit was 3.69 x 10(-12) M. The running time was less than 10 min. Selectivities were higher than 92% and the relative errors were less than 7%. The IgG concentration of serum was determined to be 3.6 mg ml(-1). This measurement differed by 8.3% from the ELISA measurement. The recoveries of IgG spiked in serum were found to be higher than 94%. This method can provide simple, fast, and selective analysis for protein detection and other immunoassay-related applications.

Integrating the QCM detection with magnetic separation for on-line analysis.

We investigate the feasibility of coupling the quartz crystal microbalance (QCM) with magnetic separation for on-line analysis. A flow cell was integrated with QCM and magnetic force for the analysis of magnetic and nonmagnetic samples. The resonant frequency change ((Delta)f) of QCM was related to the amount of deposited magnetic nanoparticles. This experiment demonstrates that QCM can be used as an on-line detector for magnetic separation. The QCM also gives a characteristic response of the binding between the streptavidin and biotin labeled on the magnetic nanoparticles. Biotin-labeled magnetic nanoparticles were flowed through a gold electrode of QCM to deposit as a matrix for selective capturing streptavidin. The resonant frequency change of QCM was proportional to the amounts of streptavidin captured by biotin. This technique can provide a simple, economic, and automatic method for on-line detection of biomarkers.

Detection of C-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles.

We report a detection method for C-reactive protein (CRP) based on competitive immunoassay using magnetic nanoparticles under magnetic fields. Functional magnetic nanoparticles were prepared and conjugated with anti-CRP for immunoassay. Magnetic nanoparticles labeled with anti-CRP were flowed through a separation channel to form depositions for selective capture of CRP under magnetic fields. Free CRP and a fixed number of CRP-labeled particles were used to compete for a limited number of anti-CRP binding sites on the magnetic nanoparticles. The deposited percentages of CRP-labeled particles at various concentrations of free CRP were determined and used as a reference plot. The determination of CRP in the unknown sample was deduced from the reference plot using the deposited percentages. The running time was less than 10 min. The CRP concentration of serum sample was linearly over the range of 1.2-310 microg/mL for deposited percentages of CRP-labeled particles. The detection limit of this method was 0.12 microg/mL which was approximately 8-fold lower than the typical clinical cutoff concentration (1 microug/mL). This method can provide a fast, simple, and sensitive way for protein detection based on competitive immunoassay using magnetic nanoparticles under magnetic fields.

Analytical and preparative applications of magnetic split-flow thin fractionation on several ion-labeled red blood cells.

BACKGROUND: Magnetic Split-flow thin (SPLITT) fractionation is a newly developed technique for separating magnetically susceptible particles. Particles with different field-induced velocities can be separated into two fractions by adjusting applied magnetic forces and flow-rates at inlets and outlets. METHODS: Magnetic particles, Dynabeads, were used to test this new approach of field-induced velocity for susceptibility determination using magnetic SF at different magnetic field intensities. Reference measurements of magnetic susceptibility were made using a superconducting quantum interference device (SQUID) magnetometer. Various ion-labeled red blood cells (RBC) were used to study susceptibility determination and throughput parameters for analytical and preparative applications of magnetic SPLITT fractionation (SF), respectively. Throughputs were studied at different sample concentrations, magnetic field intensities, and channel flow-rates. RESULTS: The susceptibilities of Dynabeads determined by SPLITT fractionation (SF) were consistent with those of reference measurement using a superconducting quantum interference device (SQUID) magnetometer. Determined susceptibilities of ion-labeled RBC were consistent within 9.6% variations at two magnetic intensities and different flow-rates. The determined susceptibilities differed by 10% from referenced measurements. The minimum difference in magnetic susceptibility required for complete separation was about 5.0 x 10(-6) [cgs]. Sample recoveries were higher than 92%. The throughput of magnetic SF was approximately 1.8 g/h using our experimental setup. CONCLUSION: Magnetic SF can provide simple and economical determination of particle susceptibility. This technique also has great potential for cell separation and related analysis. Continuous separations of ion-labeled RBC using magnetic SF were successful over 4 hours. The throughput was increased by 18 folds versus early study. Sample recoveries were 93.1 +/- 1.8% in triplicate experiments.

New method of blood typing using analytical magnetapheresis.

We report a new method of blood typing based on the agglutination of red blood cell (RBC) with serum-treated magnetic particles in analytical magnetapheresis. Blood typing of ABO was demonstrated. The agglutination patterns of RBCs are different for different blood types and can be used to determine the ABO blood typing in analytical magnetapheresis. Six samples can be tested in each run. The running time was less than 10 min. Magnetic particles were prepared in the laboratory. The amount of RBCs needed for the agglutination test was about 1.0 microl of adult blood. The blood typing of ABO was used to illustrate the capable applications of analytical magnetapheresis to nonmagnetic samples like cells without magnetic labels. Analytical magnetapheresis has a great potential for cell related analysis.

Impurity analysis of 1,4-dioxane in nonionic surfactants and cosmetics using headspace solid-phase microextraction coupled with gas chromatography and gas chromatography-mass spectrometry.

1,4-Dioxane impurity in nonionic surfactants and cosmetics were analyzed using solid-phase microextraction (SPME) coupled with gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Experimental results show that there is no significant difference using SPME-GC and SPME-GC-MS for analysis of 1,4-dioxane in three types of nonionic surfactants at the 95% confidence level. The relative standard deviation (R.S.D.) values of each analytical method were smaller than 3%. The amount of 1,4-dioxane was found to vary from 11.6 +/- 0.3 ppm to 73.5 +/- 0.5 ppm in 30% of nonionic surfactants from manufacturers in Taiwan. These methods were linear over the studied range of 3-150 ppm with correlation coefficients higher than 0.995. The recoveries of 1,4-dioxane for these nonionic surfactants following SPME were all higher than 96 +/- 1% (n = 3). The detection limits of 1,4-dioxane for these nonionic surfactants following SPME were from 0.06 ppm to 0.51 ppm. The experimentally determined level of 1,4-dioxane in cosmetics from manufacturers in Taiwan varied from 4.2 +/- 0.1 ppm to 41.1 +/- 0.6 ppm in 22% of daily used cosmetics following SPME coupled with GC and GC-MS. Conventional solvent extraction takes around 1 h for extraction and reconcentration but SPME takes only around 10 min. SPME provides better analyses of 1,4-dioxane in nonionic surfactants and cosmetics than conventional solvent extraction and head space pretreatments in term of simplicity, speed, precision, detection limit, and solvent consumption.