Distance and Microsphere Aggregation-Based DNA Detection in a Paper-Based Microfluidic Device

Abstract

In paper-based microfluidics, the simplest devices are colorimetric, giving qualitative results. However, getting quantitative data can be quite a bit more difficult. Distance-based devices provide a user-friendly means of obtaining quantitative data without the need for any additional equipment, simply by using an included ruler or calibrated markings. This article details the development of a quantitative DNA detection device that utilizes the aggregation of polystyrene microspheres to affect the distance that microspheres wick through filter paper. The microspheres are conjugated to single-stranded DNA (ssDNA) oligomers that are partially complementary to a target strand and, in the presence of the target strand, form a three-strand complex, resulting in the formation of aggregates. The higher the concentration of the target strand, the larger the aggregate, and the shorter the distance wicked by the microspheres. This behavior was investigated across a wide range of target concentrations and under different incubation times to understand aggregate formation. The device was then used to successfully detect a target strand spiked in extracted plant DNA.

Paper-Based Biosensors for the Detection of Nucleic Acids from Pathogens

“…Nanoparticle aggregation offers multiple advantages for the colorimetric detection of nucleic acids. Mainly, due to their mechanism involving target complementary oligonucleotides, they are specific, being able to differentiate double and even single nucleotide mismatches in the sequence [ 174 , 176 ], which is particularly useful for serotyping or mutant-specific detection. Given the high specificity of the aggregation phenomenon with the hybridized target sequence, they are resistant to protein interference [ 176 ] and the degree of aggregation can be correlated to the amount of target sequence, providing quantitative or semiquantitative capabilities to the device based on color intensity [ 173 , 174 , 175 , 176 ] or distance traveled by solution [ 177 , 178 ].…”

“…Mainly, due to their mechanism involving target complementary oligonucleotides, they are specific, being able to differentiate double and even single nucleotide mismatches in the sequence [ 174 , 176 ], which is particularly useful for serotyping or mutant-specific detection. Given the high specificity of the aggregation phenomenon with the hybridized target sequence, they are resistant to protein interference [ 176 ] and the degree of aggregation can be correlated to the amount of target sequence, providing quantitative or semiquantitative capabilities to the device based on color intensity [ 173 , 174 , 175 , 176 ] or distance traveled by solution [ 177 , 178 ]. Since the aggregation is dependent on the hybridization of the target to probe sequences and not the side product of another reaction, such as in detection based on pH or Mg 2+ , an amplification step is not necessary, reducing the steps and requirements for the device.…”

“…Similarly, while the nanoparticles are easily manufactured, the custom-modified oligonucleotide probes often have complex manufacturing and purification steps. The use of PNAs with positively charged lysine reduces self-aggregation and false positives, but it increases the manufacturing requirements of the ssDNA probes for each assay [ 174 , 176 ]. Although PNAs increase resistance to many interferents in complex samples, under the presence of high ionic salt concentrations such as >30 mM NaCl [ 176 ] or >200 mM MgCl 2 [ 174 ], the aggregation state of the nanoparticles did not change regardless of the addition of probes and/or target DNA.…”

A Simple Distance Paper-Based Analytical Device for the Screening of Lead in Food Matrices

“…The measurement is simple, requiring only a readout of the color distance from the nearby ruler. Distance-based detection is one of the promising semi-quantitative analysis methods with superiority to the colorimetric detection-based method [ 13 , 14 , 15 , 16 , 17 , 18 ]. Several approaches have been proposed for improving the performance of the dPADs, such as using organic solvent to enforce the opening of wax valves, which facilitates reagent mixing and incubation in distance paper-based analytical devices [ 19 ].…”

Flexible Analytical Devices for Point-of-Care Testing

“…Because this device is antibody-free and uses a smartphone to read the colorimetric signal, it enables simplified lactoferrin detection in a point-of-care format. The report by Kalish et al 5 presents distance-based detection of nucleic acids in paper-based microfluidic devices. The mechanism of this detection scheme is based on the aggregation of polystyrene microspheres conjugated with single-stranded DNA that is partially complementary to a target DNA strand.…”