Rapid colorimetric detection of Salmonella typhimuriumusing a selective filtration technique combined with antibody–magnetic nanoparticle nanocomposites

Detection of pathogenic bacteria that pose a great risk to human health requires a rapid, convenient, reliable, and sensitive detection method. In this study, we developed a selective filtration method using monoclonal antibody (MAb)–magnetic nanoparticle (MNP) nanocomposites for the rapid and sensitive colorimetric detection of Salmonella typhimurium. The method contains two key steps: the immunomagnetic separation of the bacteria using MAb–MNP nanocomposites and the filtration of the nanocomposite-bound bacteria. Color signals from the nanocomposites remaining on the membrane were measured, which reflected the amount of bacteria in test samples. Immunomagnetic capture efficiencies of 8 to 90 % for various concentrations of the pathogen (2?×?10⁴–2?×?10¹ cells) were obtained. After optimization of the method, 2?×?10¹ cells of S. typhimurium in pure culture solution was detectable as well as in artificially inoculated vegetables (100 cells/g). The method was confirmed to be highly specific to S. typhimurium without cross-reaction to other pathogenic bacteria and could be concluded within 45 min, yielding results in a shorter or similar time period as compared with recently reported antibody immobilized on magnetic-particle-based methods. This study also demonstrated direct application of MAb–MNP nanocomposites without a dissociation step of bacteria from magnetic beads in colorimetric assays in practice.     

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen–antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.     

Protein micropatterning on bifunctional organic-inorganic sol-gel hybrid materials

Active protein micropatterns and microarrays made by selective localization are popular candidates for medical diagnostics, such as biosensors, bioMEMS, and basic protein studies. In this paper, we present a simple fabrication process of thick (approximately 20 microm) protein micropatterning using capillary force lithography with bifunctional sol-gel hybrid materials. Because bifunctional sol-gel hybrid material can have both an amine function for linking with protein and a methacryl function for photocuring, proteins such as streptavidin can be immobilized directly on thick bifunctional sol-gel hybrid micropatterns. Another advantage of the bifunctional sol-gel hybrid materials is the high selective stability of the amine group on bifunctional sol-gel hybrid patterns. Because amine function is regularly contained in each siloxane oligomers, immobilizing sites for streptavidin are widely distributed on the surface of thick hybrid micropatterns. The micropatterning processes of active proteins using efficient bifunctional sol-gel hybrid materials will be useful for the development of future bioengineered systems because they can save several processing steps and reduce costs.     

A colorimetric and fluorescent chemosensor for detection of Hg2+ using counterion exchange of cationic polydiacetylene

In this study, a colorimetric and fluorescent chemosensor for mercury ions (Hg²⁺) was developed. Cationic polydiacetylene (PDA) vesicles with a quaternary ammonium cation and iodide as a counterion show a blue-to-red color transition; the color change is accompanied by a fluorescence enhancement in selective response to Hg²⁺ ions because of a perturbation of the ene–yne conjugated backbone induced by counterion exchange. It allows for selective detection of Hg²⁺ with the naked eye and the sensor is used to determine Hg²⁺ concentrations in tap water samples.     

Sequential patterning of two fluorescent streptavidins assisted by photoactivatable biotin on an aminodextran-coated surface

Sequential patterning of two fluorescent streptavidins (SAvs) was carried out using photopatterning of photoactivatable biotin (photobiotin) on an aminodextran surface, which was crucial for the minimization of non-specific binding. Photobiotin was bound by photoreaction to the amine groups of aminodextran. Water contact angle at each step during the preparation of the aminodextran surface was measured to investigate the hydrophilicity of the surfaces. The specific and nonspecific binding of a fluorescent SAv was investigated for the aminodextran surface and the amine-silane surface. The aminodextran surface almost entirely prevented nonspecific binding of a fluorescent SAv and was successfully used for sequential patterning of two fluorescent SAvs. The addition of ethanolamine (40 mM) in the photobiotin solution diminished blurring of pattern shape. To decrease pattern size, the UV light was focused on the aminodextran surface in an inverted microscope system. Under optimized conditions, two fluorescent SAvs array of approximately 25 μm size was obtained using a shadow mask of 100 μm hole size in the inverted microscope system.     

A CMOS image sensor to recognize the cardiovascular disease markers troponin I and C-reactive protein

A complementary metal oxide semiconductor (CMOS) image sensor was utilized to detect the interaction of cardiovascular disease markers, troponin I and C-reactive protein. Each marker with its respective antibodies was adsorbed to an indium nanoparticle (InNP)-coated glass substrate. Dielectric layers of antigens and antibodies bound onto and interacted on conducting InNPs. Normal room light passed through these protein-layer-bound substrates and hit the CMOS image sensor surface, and the number of photons was detected and converted into digital form. We tested this approach for real-time monitoring of cardiac disease markers based on photon count, demonstrating its low cost and its capacity to detect antigens with high sensitivity at picogram per milliliter concentration.     

A label-free, direct and noncompetitive FRET immunoassay for ochratoxin A based on intrinsic fluorescence of an antigen and antibody complex

A label-free, direct and noncompetitive homogeneous immunoassay, in which ochratoxin A (OTA) coupled with the anti-OTA antibody participates in fluorescence resonance energy transfer (FRET), was developed for the detection of OTA with great specificity and a detection limit of 1 ng mL(-1).     

Selective assembly and guiding of actomyosin using carbon nanotube network monolayer patterns

We report a new method for the selective assembly and guiding of actomyosin using carbon nanotube patterns. In this method, monolayer patterns of the single-walled carbon nanotube (swCNT) network were prepared via the self-limiting mechanism during the directed assembly process, and they were used to block the adsorption of both myosin and actin filaments on specific substrate regions. The swCNT network patterns were also used as an efficient barrier for the guiding experiments of actomyosin. This is the first result showing that inorganic nanostructures such as carbon nanotubes can be used to control the adsorption and activity of actomyosin. This strategy is advantageous over previous methods because it does not require complicated biomolecular linking processes and nonbiological nanostructures are usually more stable than biomolecular linkers.     

Colorimetric detection of PCR products of DNA from pathogenic bacterial targets based on a simultaneously amplified DNAzyme

A novel strategy was devised for colorimetric analysis of the products of the polymerase chain reaction (PCR). The method takes advantage of simultaneous amplification of a horseradish peroxidase-mimicking DNAzyme (HRPzyme) during the PCR process. It is performed using a DNA specific forward primer and a universal reverse primer containing a complementary HRPzyme sequence. The double-strand PCR products, which include the HRPzyme sequence, are treated with a mixture of hemin and TMB (3,3′,5,5′–tetramethylbenzidine) in the presence of hydrogen peroxide. The resulting HRPzyme/hemin complex then promotes a peroxidase mimicking reaction, which produces the blue colored oxidized TMB. This colorimetric method can be more easily performed than previously developed gel based detection procedures and, as a result, can be conveniently applied to the specific and sensitive colorimetric analysis of DNA sequences arising from pathogenic bacteria. The potentially broad applicability of the new method has been demonstrated by its use in the identification of the 16s rDNA of Salmonella Typhimurium. Figure

A novel strategy was devised for simple colorimetric analysis of PCR products with amplification of a horseradish peroxidase-mimicking DNAzyme(HRPzyme). This colorimetric method can be much more easily performed than previously developed gel based detection procedures and potentially broad applicability for other DNA analysis.     

Multichannel surface plasmon resonance imaging and analysis of micropatterned self-assembled monolayers and protein affinity interactions

Multichannel images of 11-mercaptoundecanoic acid and 11-mercapto-1-undecanol self-assembled monolayers together with a biospecific interferon-gamma (IFN-gamma)/anti-IFN-gamma antibody immunoaffinity interaction were observed by the two-dimensional surface plasmon resonance (2D-SPR) imaging system. With the fabricated 2D-SPR imaging system, adopting a white light source in combination with a narrow band-pass filter, sharp images were resolved, minimizing the diffraction patterns on the resulting images. Micropatterning of self-assembled monolayers was acheived by exploiting the UV photolysis of thiol bonding, instead of conventional photolithography. The line profile calibration of the image contrast with ellipsometric analysis enabled us to discriminate the change in monolayer thickness within a sub-nanometer scale. For the protein interactions on the surface, the biospecific affinity recognition reaction of IFN-gamma antigen with surface-immobilized antibody was analyzed. Through the signal amplification strategy based on the enzyme-catalyzed precipitation reaction in a sandwich-type immunoassay, biospecific antigen binding was found detectable down to a concentration of 1 ng/mL.