Mobile Platform for Multiplexed Detection and Differentiation of Disease-Specific Nucleic Acid Sequences, Using Microfluidic Loop-Mediated Isothermal Amplification and Smartphone Detection

Abstract

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a hand-held "cradle" that interfaces the phone with a silicon-based microfluidic chip embedded within a credit-card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to utilize a single 15 μL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 min. Our approach utilizes loop-mediated isothermal amplification (LAMP) reagents predeposited into distinct lanes of the microfluidic chip, which when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that when excited by appropriately selected light emitting diodes (LEDs), are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with the information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results.

An ultraportable and versatile point-of-care DNA testing platform

“…We further adopted microfluidics and iRPAS signal amplification, enabling the sensitive, selective, and multiplexed testing on a chip. In addition to that, by producing the colorimetric signals, our POCKET eliminates the need of any optical filters, making the entire platform not only ultraportable and versatile but also more cost-effective, compared with fluorescence-based systems (17). We use a smartphone not only as a readout device for quantitative analyses but also as a heater, permitting the rapid and robust point-of-care DNA amplification even at the cold environment.…”

“…Because of our combinatory and integrated approach, the POCKET system was extremely light and small, rendering the POCKET system, to the best of our knowledge, to be one of the smallest and most portable POC DNA testing devices. Compared with other 3D-printed or microfluidic chips, our POCKET system was more versatile for a broader range of possible sample sources and achieved multiplex detections with reduced reagent costs (table S6) (6,9,11,14,17,(27)(28)(29)(30)(31).…”

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus at the Point of Care

“…Since our AIOD-CRISPR assay generates strong uorescence signals at the endpoint, it is possible to record, analyze and report the detection results by taking advantage of ubiquitous smartphone technology. 41,42,43 The smartphone can be programmed to take uorescence photos, convert the images into uorescence intensity, analyze the data, and report the qualitative/semi-quantitative test results.…”

Smartphone-based pathogen diagnosis in urinary sepsis patients

“…Examples include optical and fluorescence imaging [ 20 ], microtiter assay interpretation [ 21 ], immunologic detection (e.g. microfluidic chips [ [22] , [23] , [24] ]; antibody-conjugated strips [ 25 ]) and nucleic acid detection (e.g., microfuge tubes [ 26 , 27 ]; microtiter plates [ 28 ]; microfluidic chambers [ 29 ]; microfluidic chips [ [30] , [31] , [32] , [33] , [34] , [35] , [36] ]). Although these are notable advances in terms of broadening access to sophisticated molecular diagnostics, translation to clinical utility using patient-derived samples has been limited (e.g., HIV blood samples [ 24 ], influenza throat swabs [ 25 ], Chlamydia trachomatis swabs [ 36 ]).…”