An Origami Paper-Based Device Printed with DNAzyme-Containing DNA Superstructures for Escherichia coli Detection

Abstract

Rapid detection of pathogenic bacteria is extremely important for public health and safety. Here, we describe for the first time an integrated origami paper-based analytical device (PAD) incorporating cell lysis, molecular recognition, amplification and visual detection of Escherichia coli (E. coli). The device features three components: paper for its ability to extract protein molecules nonspecifically from cells, DNA superstructures for their ability to immobilize RNA-cleaving DNAzymes (RCDs) but undergo target-induced RNA cleavage on paper, and isothermal rolling circle amplification (RCA) for its ability to amplify each cleavage event into repetitive sequence units that can be detected by naked eye. This device can achieve detection of E. coli K12 with a detection limit of as low as 103 CFU·mL−1 in a total turnaround time of 35 min. Furthermore, this device allowed the sensitive detection of E. coli in complex sample matrices such as juice and milk. Given that more specific RCDs can be evolved for diverse bacteria, the integrated PAD holds great potential for rapid, sensitive and highly selective detection of pathogenic bacteria in resource-limited settings.

Nucleic Acids Analysis

“…Benefiting from the simplicity, sensitivity and low cost, colorimetry detection technique is considered to be suitable for integrating with PADs [395]. Colorimetric assays in PADs are accomplished usually depending on the aggregation of noble nanoparticles [395,396] or the oxidation of chromogenic substrate [396][397][398][399][400]. The target-stimuli signal, color change, can be visualized by naked eyes and the detection zone images can collected by camera phone or portable scanner and then transformed into quantitative information by software [394,[401][402][403].…”

“…Various nucleic acid-mediated reactions have been exploited in PADs, including simple DNA hybridization [378,379,389,395,409] and complex reactions, such as PCR [415], RPA [398,416], RCA [396,399,417], and SDA [418,419]. The sensing interfaces are composed of oligonucleotide monolayers [379,412,420], composite multilayer [378,409] and 3D nucleic acid materials [399,410] or oligonucleotide-containing heterogeneous nanostructures [123].…”

“…Various nucleic acid-mediated reactions have been exploited in PADs, including simple DNA hybridization [378,379,389,395,409] and complex reactions, such as PCR [415], RPA [398,416], RCA [396,399,417], and SDA [418,419]. The sensing interfaces are composed of oligonucleotide monolayers [379,412,420], composite multilayer [378,409] and 3D nucleic acid materials [399,410] or oligonucleotide-containing heterogeneous nanostructures [123]. The analysts include small molecules [375,421], miRNAs [378], DNA strands [123,379,412,420], proteins [375,379,391,422], exosomes [409], cancer cells [422], papillomavirus [389], pathogens [408] and so on, implying the great potential of nucleic acid-based PADs in enormous applications.…”

Recent Advances in Microfluidic Devices for Contamination Detection and Quality Inspection of Milk

“…Various microfluidic platforms have been developed for the detection of E. coli in milk samples, including lab-on-a-chip devices combined with immunoassay [ 78 ], aptamer and fluorescence detection [ 76 ], rolling circle amplification (RCA), and fluorescence detection [ 91 ], and lab-on-paper devices based on RCA and colorimetric detection [ 105 ], colorimetric detection [ 86 , 106 ], and immunoassay [ 90 , 92 , 93 ]. Sun et al [ 105 ] developed a folding microfluidic lab-on-paper RCA device with DNAzyme-containing DNA superstructures for E. coli detection in juice and milk samples. As shown in Figure 4 a, the device first extracted the protein molecules from the cells in the sample and then used a DNA superstructure to immobilize the RNA and produce a target-induced cleavage of the DNAzymes.…”

Advanced Signal-Amplification Strategies for Paper-Based Analytical Devices: A Comprehensive Review

“…Conventionally, polymerase chain reaction (PCR) is the technique extensively used to rapidly process millions to billions of DNA samples, but it requires precise temperature control for reactions, and hence, to date has been unsuccessfully applied to PADs [ 149 , 150 ]. Alternatively, various signal amplification isothermal methods have been tried for PAD development, including rolling circle amplification (RCA) [ 147 , 151 , 152 , 153 , 154 , 155 ], loop-mediated isothermal amplification (LAMP) [ 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 ], strand-displacement amplification (SDA) [ 167 ], recombinase polymerase amplification (RPA) [ 168 , 169 , 170 , 171 ], helicase dependent amplification (HDA) [ 172 , 173 , 174 ], and nucleic acid sequence-based amplification (NASBA) [ 175 ].…”