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Electrochemical aptamer‐based (E‐AB) sensors offer a powerful and general means for analyte detection in complex samples for various applications. Paper‐based E‐AB sensors could enable portable, low‐cost, and rapid detection of a broad range of targets, but it has proven challenging to fabricate suitable three‐electrode systems on paper. Here, we demonstrate a simple, economic, and environmentally friendly strategy for fabricating aptamer‐modified paper electrochemical devices (PEDs) via ambient vacuum filtration. The material, shape, size, and thickness of the three‐electrode PED system can be fully customized. We developed aptamer‐modified PEDs that enable sensitive and specific detection of small molecules in minimally processed biosamples. The sensitivity and stability of the PEDs are comparable to E‐AB sensors based on commercial gold electrodes. We believe our strategy can lead to the development of high performance PEDs for the on‐site detection of a variety of analytes. [ABSTRACT FROM AUTHOR]

The reliable detection of pathogenic bacteria in complex biological samples using simple assays or devices remains a major challenge. Herein, we report a simple colorimetric paper device capable of providing specific and sensitive detection of Helicobacter pylori (H. pylori), a pathogen strongly linked to gastric carcinoma, gastric ulcers, and duodenal ulcers, in stool samples. The sensor molecule, an RNA‐cleaving DNAzyme obtained through in vitro selection, is activated by a protein biomarker from H. pylori. The colorimetric paper sensor, designed on the basis of the RNA‐cleaving property of the DNAzyme, is capable of sensitive detection of H. pylori in human stool samples with minimal sample processing and provides results in minutes. It remains fully functional under storage at ambient temperature for at least 130 days. This work lays a foundation for developing DNAzyme‐enabled paper‐based point‐of‐care diagnostic devices for monitoring pathogens in complex samples. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Current paper-based potentiometric ion-sensing platforms are planar devices used for clinically relevant ions. These devices, however, have not been designed for the potentiometric biosensing of proteins or small molecule analytes. A three-dimensional origami paper-based device, in which a solid-contact ion-selective electrode is integrated with an all-solid-state reference electrode, is described for the first time. The device is made by impregnation of paper with appropriate bioreceptors and reporting reagents on different zones. By folding and unfolding the paper structures, versatile potentiometric bioassays can be performed. A USB-controlled miniaturized electrochemical detector can be used for simple and in situ measurements. Using butyrylcholinesterase as a model enzyme, the device has been successfully applied to the detection of enzyme activities and organophosphate pesticides involved in the enzymatic system as inhibitors. The proposed 3D origami paper device allows the potentiometric biosensing of proteins and small molecules in a simple, portable, and cost-effective way. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

φ29 DNA polymerase (Polφ29) is capable of synthesizing long‐chain single‐stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote "micrometer‐sized DNA track" (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer‐binding domain at its 3′ end and ≈1000 repeating sequence units at its 5′ end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5‐time faster than it does a related ssCDT. We use this to design a simple all‐in‐one printed paper device for rapid and sensitive detection of microRNA let‐7. This paper sensor is capable of detecting 1 pM let‐7a in 10 minutes. [ABSTRACT FROM AUTHOR]

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world. [ABSTRACT FROM AUTHOR]