Self-Assembled Functional DNA Superstructures as High-Density and Versatile Recognition Elements for Printed Paper Sensors.

Micrometer-sized functional nucleic acid (FNA) superstructures (denoted as 3D DNA) were examined as a unique class of biorecognition elements to produce highly functional bioactive paper surfaces. 3D DNA containing repeating sequences of either a DNA aptamer or DNAzyme was created from long-chain products of rolling circle amplification followed by salt aging. The resulting 3D DNA retained its original spherical shape upon inkjet printing and adhered strongly to the paper surface via physisorption. 3D DNA paper sensors showed resistance to degradation by nucleases, suppressed nonspecific protein adsorption, and provided a much higher surface density of functional DNA relative to monomeric FNAs, making such species ideally suited for development of paper-based biosensors.

Print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens.

Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10-50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ.

A Printed Multicomponent Paper Sensor for Bacterial Detection.

We present a simple all-in-one paper-based sensor for E. coli detection using a composite ink made of a fluorogenic DNAzyme probe for bacterial recognition and signal generation, lysozyme that lyses whole bacterial cells, and pullulan/trehalose sugars that stabilize printed bioactive molecules. The paper sensor is capable of producing a fluorescence signal as a readout within 5 minutes upon contacting E. coli, can achieve a limit of detection of 100 cells/mL, in a variety of sample matrixes, without sample enrichment, and remains stable for at least 6 months when stored at ambient temperature. Therefore, this simple paper sensor provides rapid bacterial testing on site, and can be shipped and stored under ambient conditions to benefit users living in resource-limited regions.

Automating multi-step paper-based assays using integrated layering of reagents.

We describe a versatile and simple method to perform sequential reactions on paper analytical devices by stacking dry pullulan films on paper, where each film contains one or more reagents or acts as a delay layer. Exposing the films to an aqueous solution of the analyte leads to sequential dissolution of the films in a temporally controlled manner followed by diffusive mixing of the reagents, so that sequential reactions can be performed. The films can be easily arranged for lateral flow assays or for spot tests (reactions take place sequentially in the z-direction). We have tested the general feasibility of the approach using three different model systems to demonstrate different capabilities: 1) pH ramping from low to high and high to low to demonstrate timing control; 2) rapid ready-to-use two-step Simon's assays on paper for detection of drugs of abuse utilizing a 2-layer stack containing two different reagents to demonstrate the ability to perform assays in the z-direction; and 3) sequential cell lysing and colorimetric detection of an intracellular bacterial enzyme, to demonstrate the ability of the method to perform sample preparation and analysis in the form of a spot assay. Overall, these studies demonstrate the potential of stacked pullulan films as useful components to enable multi-step assays on simple paper-based devices.

Target-Induced and Equipment-Free DNA Amplification with a Simple Paper Device.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.

Simple and ultrastable all-inclusive pullulan tablets for challenging bioassays.

Many biodetection systems employ labile enzymes and substrates that need special care, making it hard to routinely use them for point-of-care or field applications. In this work we provide a simple solution to this challenging problem through the creation of all-inclusive pullulan assay tablets. The proposed tablet system not only enhances the long-term stability of both enzymes and organic substrates, but also simplifies the assay procedure. The enhanced stability is attributed to two factors: the restriction of the molecular motion of proteins and impermeability to molecular oxygen afforded by the tables. These tablets dissolve rapidly upon addition to testing samples, making the test very easy to perform. Using the ATP-detecting luciferase-luciferin system as an example, we show that the tablet-based assay can achieve highly sensitive detection of ATP in biological samples and that the activity of the assay tablets remains unchanged for over a month at room temperature.

Design Rules for Fluorocarbon-Free Omniphobic Solvent Barriers in Paper-Based Devices.

The utility of hydrophobic wax barriers in paper-based lateral flow and multiwell devices for containment of aqueous solvents was extended to organic solvents and challenging aqueous surfactant solutions by preparation of a three layer barrier, consisting of internal pullulan impregnated paper barriers surrounded by external wax barriers. When paper impregnated with pullulan solution dries, the polymer forms solvent blocking lenses in the paper structure. Lens formation was illustrated by forming pullulan lenses in glass capillaries. The lens shapes were less curved compared to the predictions of a model based upon minimizing surface area. For barriers on Whatman # 1 filter paper, the pullulan molecular weight must be greater than ∼70 kDa, the mass fraction of pullulan in the barrier zone must be at least 32%, and there are restrictions on the minimum width of the pullulan impregnated zone.

Printed paper sensors for serum lactate dehydrogenase using pullulan-based inks to immobilize reagents.

In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

Amine-phenyl multi-component gradient stationary phases.

Continuous multi-component gradients in amine and phenyl groups were fabricated using controlled rate infusion (CRI). Solutions prepared from either 3-aminopropyltriethoxysilane (APTEOS) or phenyltrimethoxysilane (PTMOS) were infused, in a sequential fashion, at a controlled rate into an empty graduated cylinder housing a vertically aligned thin layer chromatography (TLC) plate. The hydrolyzed precursors reacted with an abundance of silanol (SiOH) groups on the TLC plates, covalently attaching the functionalized silane to its surface. The extent of modification by phenyl and amine was determined by the kinetics of each reaction and the exposure time at each point along the TLC plate. The local concentrations of phenyl and amine were measured using diffuse reflectance spectroscopy and X-ray photoelectron spectroscopy, respectively. The profile of the multi-component gradients strongly depended on the order of infusion, the direction of the gradient and the presence of available surface silanol groups. A slightly higher amount of phenyl can be deposited on the TLC plate by first modifying its surface with amine groups as they serve as a catalyst, enhancing condensation. Separation of water- and fat-soluble vitamins and the control of retention factors were demonstrated on the multi-component gradient TLC plates. Uniformly modified and single-component TLC plates gave different separations compared to the multi-component gradient plates. The retention factors of the individual vitamins depended on the order of surface modification, the spotting end, and whether the multi-component gradients align or oppose each other.

Chelation gradients for investigation of metal ion binding at silica surfaces.

Centimeter-long surface gradients in bi- and tridentate chelating agents have been formed via controlled rate infusion, and the coordination of Cu(2+) and Zn(2+) to these surfaces has been examined as a function of distance by X-ray photoelectron spectroscopy (XPS). 3-(Trimethoxysilylpropyl)ethylenediamine and 3-(trimethoxysilylpropyl)diethylenetriamine were used as precursor silanes to form the chelation gradients. When the gradients were exposed to a metal ion solution, a series of coordination complexes formed along the length of the substrate. For both chelating agents at the three different concentrations studied, the amine content gradually increased from top to bottom as expected for a surface chemical gradient. While the Cu 2p peak area had nearly the same profile as nitrogen, the Zn 2p peak area did not and exhibited a plateau along much of the gradient. The normalized nitrogen-to-metal peak area ratio (N/M) was found to be highly dependent on the type of ligand, its surface concentration, and the type of metal ion. For Cu(2+), the N/M ratio ranged from 8 to 11 on the diamine gradient and was ∼4 on the triamine gradient, while for Zn(2+), the N/M ratio was 4-8 on diamine and 5-7 on triamine gradients. The extent of protonation of amine groups was higher for the diamine gradients, which could lead to an increased N/M ratio. Both 1:1 and 1:2 ligand/metal complexes along with dinuclear complexes are proposed to form, with their relative amounts dependent on the ligand, ligand density, and metal ion. Collectively, the methods and results described herein represent a new approach to study metal ion binding and coordination on surfaces, which is especially important to the extraction, preconcentration, and separation of metal ions.

Pullulan encapsulation of labile biomolecules to give stable bioassay tablets.

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components.

Fabrication of surface charge gradients in open-tubular capillaries and their characterization by spatially resolved pulsed streaming potential measurements.

Surface charge gradients have been formed on the inside surface of 75 µm i.d. silica capillaries via controlled rate infusion using 3-aminopropyltriethoxysilane as the reactive precursor. These 400 mm length gradients have been characterized using spatially resolved streaming potential measurements, from which the zeta potential as a function of distance was determined. The gradient capillaries exhibited a gradual variation in zeta potential from top to bottom, whereas uniformly modified and as-received capillaries were relatively homogeneous along their length. For a gradient prepared with a relatively high concentration of aminosilane, the zeta potential changed over 60 mV from one end of the capillary to the other, yielding a variation in the magnitude of the apparent surface charge of ~7 fold. By changing the concentration of the aminoalkoxysilane and/or the rate of infusion, both the value of the zeta potential (and hence surface charge) and its spatial profile (i.e., rate of change with distance) could be manipulated.

Aminoalkoxysilane reactivity in surface amine gradients prepared by controlled-rate infusion.

The reactivity of a series of substituted aminoalkoxysilanes for surface amine gradient formation has been studied using a newly developed time-based exposure method termed controlled-rate infusion (CRI). The aminoalkoxysilanes used include those that contain primary, secondary, and tertiary monoamines as well as more than one amine group (diamine and triamine). X-ray photoelectron spectroscopy (XPS) was used to confirm the presence of a gradient in each case and to acquire detailed information on gradient composition from which kinetic data were obtained. The total area under the N 1s XPS spectra allows for the extent of amine modification to be quantitatively assessed along each gradient. The N 1s peaks actually appear as doublets, providing additional data on the level of protonation and, hence, amine basicity on the dry surface. The degree of protonation showed an interesting trend toward smaller values running from top to bottom along gradients incorporating the most basic amines. The gradient profiles, including initial steepness and extent of saturation, were shown to be highly dependent on the aminoalkoxysilane precursor employed. The highest levels of modification were achieved for the diamine and primary monoamine precursors while the more hindered amines produced lower levels of surface modification and took longer for saturation to be achieved. By fitting the gradient data to a simple first-order kinetic model, rate constants for the condensation reaction between each aminosilane and accessible surface silanol groups were obtained. The rate constants follow the trend: triamine ~ diamine > monoamine and primary > secondary > tertiary, indicating kinetic factors also play an important role in controlling surface modification. The presence of more than one amine group on the silane is concluded to enhance the rate of condensation to the surface silanol groups, while the more hindered secondary and tertiary amines slow condensation. Collectively, the results provide valuable new data on how the number of amine groups, degree of substitution, and steric hindrance influence silane reactivity with silica surfaces, amine surface coverage, and basicity along the gradient profile.

Continuous stationary phase gradients for planar chromatographic media.

A simple, elegant method for the formation of a continuous stationary phase gradient for use in chromatographic separations is described. Its applicability to separation science is demonstrated using thin-layer chromatography as a test case. Gradient stationary phases were formed on activated High Performance Thin-Layer Chromatography (HP-TLC) plates using a newly developed methodology termed "controlled rate infusion". Specifically, the SiOH groups on the activated HP-TLC plates were reacted with 3-aminopropyltriethoxysilane (APTEOS) in a time dependent fashion by using a programmable syringe pump to control the rate of APTEOS infusion into the deposition reservoir. The shape (profile) of the gradient was controlled by the rate of infusion and imaged by taking advantage of the concentration-dependent color formation reaction between amine groups and ninhydrin. The advantages of such gradients in optimizing the retention and separation of various components in different mixtures were illustrated using mixtures of (1) four weak acids and bases and (2) three widely used over-the-counter drugs. The separation of the individual components on the gradient stationary phase was clearly improved relative to those on either traditional normal-phase TLC plates or uniformly amine-modified TLC plates. Precise control over component retention and separation was also demonstrated by strategically modifying the steepness of the gradient.

Profile control in surface amine gradients prepared by controlled-rate infusion.

Surface amine gradients that exhibit a wide variety of profiles, including those that incorporate spatially distinct regions having steep and gradual variations in chemical functionality, have been prepared by the sol-gel process using a controlled-rate infusion method. In this work, a substrate that incorporates dimethyl and Si-OH groups is temporally modified with an aminoalkoxysilane (NH(2)(CH(2))(3)Si(OC(2)H(5))(3)) to build a gradient film for which the amine content changes over a 10-20 mm distance. Both X-ray photoelectron spectroscopy (XPS) and contact angle measurements confirm the presence of a chemical gradient across the surface of the film. As expected, a greater density of amine functionalities and lower contact angle were found at the bottom of the gradient relative to the top. The local steepness of the gradient was systematically controlled by changing the rate of infusion. Fast rates of infusion created gradient surfaces where the amine content changed slowly along the surface and never reached saturation, whereas slow rates of infusion formed a surface exhibiting a steep rise in amine content followed by saturation. The steepness of the gradient was also changed at predefined positions along its length by programming the rate of infusion. Gradients prepared using six-step, three-step, and two-step programmed infusion rates are shown. The data fit nicely to a kinetic model that assumes first-order kinetics. The ability to manipulate the gradient profile is particularly vital for applications that rely on mass transport and/or those that require spatial control of gradient properties.