Search

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.

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.

A potentially universal approach is presented for enzyme attachment to cellulose that significantly enhances enzyme stability while retaining high activity, and involves no chemical functionalization of cellulose. Bovine serum albumin (BSA) was interlocked in cellulose to form a protein-friendly surface (named BSA-Paper), while also providing COOH and NH2 groups for subsequent attachment of enzymes. The desired enzyme is then mixed with additional BSA and interlocked on BSA-Paper. The second BSA layer dilutes and crosslinks the enzyme for improved stability. Laccase was tested as a model enzyme for interlocking on BSA-Paper, and was found to retain over 100 % activity and was 240 times more stable at 25 °C (half life=180 d) than laccase. This new approach was also tested with a few other enzymes with encouraging results, thus providing a potentially universal method for stabilization of enzymes on cellulose with retention of high activities.

We report a general anion sensing platform based on a portable and cost-effective ion-selective optode and a smartphone detector equipped with a color analysis app. In contrast to traditional anion-selective optodes using a hydrophobic polymer and/or plasticizer to dissolve hydrophobic sensing elements, the new optode relies on hydrophilic cellulose paper. The anion ionophore and a lipophilic pH indicator are inkjet-printed and adsorbed on paper and form a “dry” hydrophobic sensing layer. Porous cellulose sheets also allow the sensing site to be modified with dried buffer that prevents any sample pH dependence of the observed color change. A highly selective fluoride optode using an Al(III)-porphyrin ionophore is examined as an initial example of this new anion sensing platform for measurements of fluoride levels in drinking water samples. In addition to Lewis acid-base recognition, hydrogen bonding recognition is also shown to be compatible with this sensing platform.

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell-based assays. Solvent-repelling barriers made of Teflon-impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow-through syntheses on paper. The confinement and flow-through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60 % purity for the majority of the peptides (>95 % yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell-based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation.

Paper is one of the most frequently tested surfaces for the detection of latent fingermarks in criminal and terroristrelated investigations. Despite the plethora of modern physical and chemical fingerprint-detection techniques, a considerable portion of the latent fingermarks still escape detection. A plausible explanation is the remarkable difference in sweat composition between individual persons. A potential remedy to this problem could be achieved by reversing the roles, that is, developing a fingerprint-detection technique in which paper serves as the substrate for the interaction with the reagent, while the latent fingermarks will serve as a mask. In this work, “negative” fingermarks have been developed on paper, even after soaking in water, by the application of a new bifunctional reagent attached to gold nanoparticles (AuNPs), and then a silver physical developer (Ag-PD). The bifunctional reagent is composed of an active head, that is, a polar group with high affinity to cellulose, attached by a long chain to an active tail containing a sulfur group, which can stabilize AuNPs. Through the active head, the AuNPs, which are stabilized by the active tail, adhere preferentially to the paper cellulose rather than to the fingerprint material, to which they conventionally bind. Consequently, Ag-PD, which normally develops sebaceous fingermarks by precipitating dark silver on the sebaceous material, precipitate preferentially on the gold-coated areas giving rise to the appearance of uncoloured ridge detail on a dark background. In this competing process, the paper itself serves as the substrate, whereas the fingermarks serve as a mask. This process may increase the overall yield of developed fingermarks as it bypasses the issue of the remarkable differences in sweat composition between individual persons. Functionalized nanoparticles have drawn great interest during the past decade not only in potential applications as biomedical, electronic, catalytic, and optical materials, but also in forensic science, as visualizing reagents for latent fingermarks. Accumulative results on a large number of paper items such as used checks, all of which are supposed to bear latent fingemarks, have shown that over 50% escape detection of identifiable marks. Such observations have led to intensive research into more-sensitive fingerprint detection techniques. Paper that has been wetted is a particular challenge since the amino acids, which are the main substrate for chemical enhancement of latent fingermarks, are dissolved and removed by the water. A silver physical developer (Ag-PD), comprising an aqueous solution of silver nanoparticles (AgNPs) stabilized by cationic surfactants, is required to achieve satisfactory development of such fingermarks. Silver slowly deposits on the water-insoluble components of the sweat, forming dark grey to black impressions (Figure 1). Although the technique is quite sensitive, it suffers from several inconveniences, including complexity, lack of reproducibility, solution instability, and often poor contrast. 6] As a result, many forensic laboratories refrain from using this technique on a routine basis. Latent fingermark enhancement by gold nanoparticles stabilized by citrate ions in aqueous medium, and then a modified Ag-PD, is currently used in a process known as colloidal gold or multimetal deposition (MMD). AuNPs adhere to the fingermark residue and catalyze the precipitation of metallic silver from the Ag-PD solution. The gold adherence to the fingermark material is explained by an ionic interaction between the negatively charged gold colloids and the positively charged components of the fingermark residue at low pH. In a modification known as SMD (singlemetal deposition), the enhancement of gold colloids by precipitation of silver, was replaced by gold-based growth of the nanoparticles. Several other fingerprint techniques that are also based on nanochemical processes, have been suggested recently; all of these suggestions involve the adherence of nanoparticles to the fingerprint material. 3, 8] Previously we showed that good quality fingermarks were developed by treatment with an organic solution of hydrophobic AuNPs stabilized by long chain thiols, and then with Figure 1. Sebaceous fingermark developed by Ag-PD.

Here we report a self-powered origami paper analytical device (oPAD) that uses an aptamer to recognize an analyte, a glucose oxidase (GOx) tag to modify the relative concentrations of an electroactive redox couple, and a digital multimeter (DMM) to transduce the result of the assay. The sensor is self-powered in that it self-generates an electrical signal so that the read-out process is similar to testing a battery. The principle of the sensor is illustrated in Scheme 1. Briefly, the device is printed on a single piece of paper, folded into a three-dimensional (3D) configuration, and then laminated in plastic. An aliquot of sample is loaded at the inlet, split into two channels, and then directed to microbeads entrapped within the channels. In one channel, an aptamer immobilized on microbeads binds to the target and releases a GOx-labeled DNA strand that flows downstream. No aptamer is present on the microbeads in the other channel, which is used as a control. The split fluids terminate in an hour-glass-shaped, two-compartment electrochemical cell. The waist of the hour glass serves as a salt bridge between the two half cells. In one of the half cells, GOx catalyzes the oxidation of glucose, which in turn results in conversion of Fe(CN)63− to Fe(CN)64−.The difference in concentrations of Fe(CN)63− and Fe(CN)64− in the sensing half cell and control half cell results in a voltage that is used to charge a capacitor. When the switch (lower part of Scheme 1) is closed, the capacitor discharges through the DMM. The capacitor provides a high instantaneous current, in effect an amplified current, and hence a higher sensitivity than a direct current measurement.