Zwitterion-Coated Colloidal Magnetic Nanoparticle Clusters for Reduced Nonspecific Adsorption of Biomolecules.

This paper demonstrates fabrication of silica-shell-coated magnetic nanoparticle clusters (SMNCs) and subsequent surface engineering of SMNCs to produce surface-modified SMNCs that have zwitterionic and primary amine ligands (SMNC-ZW/Am). SMNC-ZW/Am was passivated by zwitterionic ligands for improved colloidal stability and reduced nonspecific adsorption and by primary amine ligands for facilitated conjugation with biomolecules. Hydrodynamic (HD) size and zeta potential of SMNC-ZW/Am could be flexibly tuned by controlling the relative amounts of zwitterionic and primary amine ligands. SMNC-ZW/Am had higher colloidal stability in high salt concentration and broad pH range than did bare SMNC. Nonspecific adsorption with biomolecules onto SMNC-ZW/Am surface was significantly suppressed by the zwitterionic ligands. The facile bioconjugation capability of SWNC-ZW/Am enabled conjugation of biotin and antibody to the SWNC-ZW/Am surface. Biomolecule-conjugated SMNC-ZW/Am showed specific binding affinity to streptavidin and Salmonella bacteria, with reduced nonspecific adsorption; therefore, SWMC-ZW/Am has potential use as an antifouling nanosubstrate for separation and bioanalysis.

Immunomagnetic separation and size-based detection of Escherichia coli O157 at the meniscus of a membrane strip.

We developed a facile method for the detection of pathogenic bacteria using gold-coated magnetic nanoparticle clusters (Au@MNCs) and porous nitrocellulose strips. Au@MNCs were synthesized and functionalized with half-fragments of Escherichia coli O157 antibodies. After the nanoparticles were used to capture E. coli O157 in milk and dispersed in a buffer solution, one end of a test strip was dipped into the solution. Due to the size difference between the E. coli-Au@MNC complexes (approximately 1 µm) and free Au@MNCs (approximately 180 nm), only E. coli-Au@MNC complexes accumulated at the meniscus of the test strip and induced a color change. The color intensity of the meniscus was proportional to the E. coli concentration, and the detection limit for E. coli in milk was 103 CFU mL-1 by the naked eye. The presence of E. coli-Au@MNC complexes at the meniscus was confirmed using a real-time PCR assay. The developed method was highly selective for E. coli when compared with Salmonella typhimurium, Listeria monocytogenes, and Staphylococcus aureus.

Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead-based applications.

This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead-encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin-biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules.

Direct immobilization of antibodies on Zn-doped Fe3O4 nanoclusters for detection of pathogenic bacteria.

Zinc-doped magnetic nanoclusters (Zn-MNCs) were synthesized and used to detect pathogenic bacteria in milk. Hydrothermally synthesized Zn-MNCs exhibited stronger magnetic properties than pure MNCs, which facilitated the magnetic separation from the sample using a permanent magnet. The presence of accessible Zn sites allows the direct immobilization of half-fragmented antibodies over Zn-MNCs through strong ZnS bonds and prevents the tedious multiple steps of molecular functionalization or coating with costly noble metals prior to conjugation with an antibody. After the capture and magnetic separation of Salmonella in milk using the antibody-functionalized Zn-MNCs, the concentration of bacteria was determined with a portable ATP luminometer and the detection limit was found to be 10 CFU/mL.

Silicon nanowire biosensors for detection of cardiac troponin I (cTnI) with high sensitivity.

We have demonstrated highly sensitive and label-free detection of cardiac troponin I (cTnI), a biomarker for diagnosis of acute myocardial infarction, using silicon nanowire field-effect transistors. A honeycomb-like structure is utilized for nanowire configuration to offer improved electrical performance and increased sensing area. The fabricated devices show n-type behavior with a relatively high ON-OFF current ratio, small sub-threshold swing and low gate leakage current. Monoclonal antibodies for cTnI were covalently immobilized on the nanowire surface and the attachment of antibodies is clearly visualized by atomic force microscope. The sensitivity with various concentrations of buffer solution was also investigated in order to determine the optimal buffer condition. The devices exhibit highest sensitivity under buffer solutions with low ion concentration. In addition, the detection limit of the sensor is as low as ~5 pg/mL, the lowest reported in the literature to date and nearly an order of magnitude smaller than the suggested threshold limit. The fabricated devices demonstrate a good selectivity for detecting cTnI.

Colorimetric detection of pathogenic bacteria using platinum-coated magnetic nanoparticle clusters and magnetophoretic chromatography.

A colorimetric method that uses platinum-coated magnetic nanoparticle clusters (Pt/MNCs) and magnetophoretic chromatography is developed to detect pathogenic bacteria. Half-fragments of monoclonal Escherichia coli O157:H7 (EC) antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. After magnetic separation of free Pt/MNCs and Pt/MNC-EC complexes from the milk, a precision pipette was used to imbibe the E. coli-containing solution, then a viscous polyethylene glycol solution. Due to difference in viscosities, the solutions separate into two liquid layers inside the pipette tip. The Pt/MNC-EC complexes were separated from the free Pt/MNCs by applying an external magnetic field, then added to a tetramethylbenzidine (TMB) solution. Catalytic oxidation of TMB by Pt produced color changes of the solution, which enabled identification of the presence of 10 cfu mL(-1) E. coli bacteria with the naked eye. The total assay time including separation, binding and detection was 30 min.

Facile detection of Troponin I using dendritic platinum nanoparticles and capillary tube indicators.

A facile method was developed for the detection of Troponin I (TnI) using dendritic platinum nanoparticles and capillary tube indicators. Dendritic platinum nanoparticles were functionalized with TnI antibodies, which were used to capture TnI in human serum. The captured TnI was conjugated to the inner surface of a glass vial, to which a hydrogen peroxide (H2O2) solution was added. After the glass vial was sealed with a screw cap containing a silicon septum, a capillary tube containing a drop of ink was inserted through the septum. The catalytic dissociation of H2O2 to water and oxygen increased the pressure inside the glass vial and raised the ink level in the capillary tube. The ink level increased with the platinum nanoparticle concentration, which is proportional to the TnI concentration. The sensitivity of this assay for TnI in human serum after a 5 min dissociation reaction, detected with the naked eye, was 0.1 ng/mL, which was better than the sensitivity of the conventional colorimetric method using the TMB oxidation reaction under the same experimental conditions. A control experiment using alpha-fetoprotein, interleukin-5, and C-reactive protein revealed that the developed method was highly selective for the detection of TnI.

3D-printed microfluidic device for the detection of pathogenic bacteria using size-based separation in helical channel with trapezoid cross-section.

A facile method has been developed to detect pathogenic bacteria using magnetic nanoparticle clusters (MNCs) and a 3D-printed helical microchannel. Antibody-functionalized MNCs were used to capture E. coli (EC) bacteria in milk, and the free MNCs and MNC-EC complexes were separated from the milk using a permanent magnet. The free MNCs and MNC-EC complexes were dispersed in a buffer solution, then the solution was injected into a helical microchannel device with or without a sheath flow. The MNC-EC complexes were separated from the free MNCs via the Dean drag force and lift force, and the separation was facilitated in the presence of a sheath flow. The concentration of the E. coli bacteria was determined using a light absorption spectrometer, and the limit of detection was found to be 10 cfu/mL in buffer solution and 100 cfu/mL in milk.

Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay.

We developed a novel 3D immunomagnetic flow assay for the rapid detection of pathogenic bacteria in a large-volume food sample. Antibody-functionalized magnetic nanoparticle clusters (AbMNCs) were magnetically immobilized on the surfaces of a 3D-printed cylindrical microchannel. The injection of a Salmonella-spiked sample solution into the microchannel produced instant binding between the AbMNCs and the Salmonella bacteria due to their efficient collisions. Nearly perfect capture of the AbMNCs and AbMNCs-Salmonella complexes was achieved under a high flow rate by stacking permanent magnets with spacers inside the cylindrical separator to maximize the magnetic force. The concentration of the bacteria in solution was determined using ATP luminescence measurements. The detection limit was better than 10 cfu/mL, and the overall assay time, including the binding, rinsing, and detection steps for a 10 mL sample took less than 3 min. To our knowledge, the 3D immunomagnetic flow assay described here provides the fastest high-sensitivity, high-capacity method for the detection of pathogenic bacteria.

Facile and sensitive method for detecting cardiac markers using ubiquitous pH meters.

A sensitive and easy method was developed for the detection of the cardiac marker troponin I using magnetic immunoassay and ubiquitous pH meters. Monoclonal antibody-functionalized Fe3O4 magnetic nanoparticle clusters (MNCs) were synthesized to capture troponin in human serum, and MNC-troponin complexes were magnetically isolated using a permanent magnet. These complexes were subsequently conjugated to polyclonal antibody-functionalized acetylcholinesterase (AchE) and dispersed in acetylcholine (Ach) solution. As the Ach was hydrolyzed to choline and acetic acid, the pH of the solution decreased, and the resulting pH change was measured in real time using a pH meter. The sensitivity of detection of this assay was found to be 10 pg/mL of troponin in human serum after 10 min of the hydrolysis reaction. Further, the pH change could be determined with the naked eye from the color change of a pH indicator strip.

Magnetophoretic chromatography for the detection of pathogenic bacteria with the naked eye.

A facile and sensitive analytical method that uses gold-coated magnetic nanoparticle clusters (Au/MNCs) and magnetophoretic chromatography with a precision pipet has been developed for the detection of Salmonella bacteria. Antibody-conjugated Au/MNCs are used to capture the Salmonella bacteria in milk and are then separated from the milk by applying an external magnetic field. The Salmonella-containing solution is sucked into a precision pipet tip to which a viscous polymer solution is then added. Once the magnetophoretic chromatography process has been carried out for 10 min, the presence of 100 cfu/mL Salmonella bacteria can be detected with the naked eye because the bacteria have become concentrated at the narrow pipet tip. The performance of this method was evaluated by using dynamic light scattering and light absorption spectroscopy.

A rapid and facile signal enhancement method for microcantilever-based immunoassays using the agglomeration of ferromagnetic nanoparticles.

A rapid and facile signal enhancement method for detecting alpha-fetoprotein (AFP) was developed using the magnetic agglomeration of ferromagnetic nanoparticles and microcantilever sensors. The resonance frequency and deflection of the cantilevers were found to be more than 10-fold greater than that before physical agglomeration of the free nanoparticles around the magnetized nanoparticles.

A facile and sensitive detection of pathogenic bacteria using magnetic nanoparticles and optical nanocrystal probes.

We report a facile and sensitive analytical method for the detection of pathogenic bacteria. Salmonella bacteria in milk were captured by antibody-conjugated magnetic nanoparticles (MNPs) and separated from analyte samples by applying an external magnetic field. The MNP-Salmonella complexes were re-dispersed in a buffer solution then exposed to antibody-immobilized TiO(2) nanocrystals (TNs), which absorb UV light. After magnetically separating the MNP-Salmonella-TN complexes from solution, the UV-Vis absorption spectrum of the unbound TN solution was obtained. Because the light absorption intensity was reversely proportional to the Salmonella concentration, the assay exhibited high sensitivity toward low concentrations of Salmonella bacteria. The detection limit of Salmonella in milk was found to be more than 100 cfu mL(-1).

Highly sensitive diagnostic assay for the detection of protein biomarkers using microresonators and multifunctional nanoparticles.

We developed a novel gravimetric immunoassay for sensitive detection of multiple protein biomarkers using silicon microcantilever arrays and multifunctional hybrid nanoparticles. Magnetic-photocatalytic hybrid nanoparticles with a highly crystalline TiO(2) shell were synthesized using a solvothermal reaction without a calcination process. After functionalizing the hybrid nanoparticles and silicon cantilevers with antibodies, the nanoparticles were used to magnetically separate target biomarkers from human serum. Frequency changes of the microcantilevers due to the binding of the nanoparticles were measured using a dip-and-dry method. Frequency changes were further amplified using a photocatalytic silver reduction reaction. Several biomarkers, including interleukin-6, interferon-γ, and alpha-fetoprotein, were selectively detected using arrays of eight silicon microcantilevers. The detection limit of this assay was ∼0.1 pg/mL, which is superior to the clinical threshold of the biomarkers.

A facile and sensitive immunoassay for the detection of alpha-fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering.

A facile and sensitive immunoassay protocol for the detection of alpha-fetoprotein (AFP) was developed using gold-coated iron oxide magnetic nanoclusters and dynamic light scattering (DLS) methods. The increase in the average particle size due to AFP-mediated aggregation was measured using DLS, and the detection limit was better than 0.01 ng mL(-1).