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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.

With over 4.8 million Canadians suffering from chronic airway diseases, respiratory exacerbations are currently the leading cause of hospitalization in Canada. In cases of bacterial infection, neutrophil cell density increases from ∼10 million cells per gram to over 15 million cells per gram. As sputum is a direct discharge from the primarily affected areas of respiratory diseases, quantification of granulocytes (including neutrophils) can be used to effectively determine a course of patient treatment. Unfortunately this quantification is currently limited to labour-intensive and time-consuming cell counts. In the present study, we describe a simple one-step lateral flow test (LFT) that can semi-quantitatively determine myeloperoxidase (MPO), a biomarker found in neutrophils, in minimally-processed sputum samples. This point-of-need (PON) diagnostic device provides positive results observable to the naked eye after 15 minutes. 37 human sputum samples were quantified for MPO using the developed LFT and compared to neutrophil levels quantified through traditional cell counting. A trend between sputum MPO concentration and total neutrophils was observed, suggesting that the LFT has the potential to replace cell counting for neutrophil approximation to aid in directing therapies quickly at the point of need.

We report a paper-based aptasensor platform that uses two reaction zones and a connecting bridge along with printed multifunctional bio/nano materials to achieve molecular recognition and signal amplification. Upon addition of analyte to the first zone, a fluorescently labelled DNA or RNA aptamer is desorbed from printed graphene oxide, rapidly producing an initial fluorescence signal. The released aptamer then flows to the second zone where it reacts with printed reagents to initiate rolling circle amplification, generating DNA amplicons containing a peroxidase-mimicking DNAzyme, which produces a colorimetric readout that can be read in an equipment-free manner or with a smartphone. The sensor was demonstrated using an RNA aptamer for adenosine triphosphate (a bacterial marker) and a DNA aptamer for glutamate dehydrogenase (Clostridium difficile marker) with excellent sensitivity and specificity. These targets could be detected in spiked serum or feacal samples, demonstrating the potential for testing clinical samples.

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.