Fully Integrated Microfluidic Biosensor with Finger Actuation for the Ultrasensitive Detection of Escherichia coli O157:H7

Abstract

A portable microfluidic biosensor was developed for the detection of E. coli O157:H7 using finger actuation. The chip was assembled with three functional zones, immunomagnetic separation, nucleic acid extraction and purification, and signal detection. First, antibody-modified magnetic nanoparticles (MNPs) were used to separate the target bacteria from the sample. The captured bacteria were then lysed and silica-coated MNPs were used to absorb DNA, followed by washing and eluting to obtain purified DNA. The obtained DNA was subjected to amplification and fluorescence detection based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeat-associated protein/Cas12a reaction. The fluorescence images were collected and analyzed using a smartphone app under a 3D-printed detection device. It could quantitatively detect E. coli O157:H7 from 10 2 to 10 8 CFU/mL in 2.5 h with a limit of detection (LOD) of 10 CFU/mL. The recovery rate ranged from 104 to 120%. Overall, the biosensor realizes "sample-in and answer-out" assay for E. coli O157:H7 and eliminates the need for external pumps and skilled personnel.

Coupling CRISPR/Cas12a and Recombinase Polymerase Amplification on a Stand-Alone Microfluidics Platform for Fast and Parallel Nucleic Acid Detection

“…For POC applications, it is ideal to achieve a samplein-answer-out format. 33 To further improve the performance of our platform, an automatic system was developed, which includes a modified microfluidic device (allows the preloading of all the reagents without potential cross-contamination, Figure S13a) and a fluidic controlling system (Figure S3b). A micropump controlled by a Digital Display Delay Relay (DDDR) card was used to automatically control the reaction process after all the reagents were loaded.…”

“…Another significant advantage of CRISPR-Dx systems is the compatibility of their reaction temperatures with those of typical isothermal amplification methods such as recombinase polymerase amplification (RPA) − and loop-mediated isothermal amplification (LAMP) . By leveraging this feature, a few studies have demonstrated the possibility of detecting various pathogens with a simple equipment setup. − Zhang’s group reported portable platforms of SHERLOCK and SHERLOCKv2 for the detection of single or multiple nucleic acid targets. , Broughton et al demonstrated a CRISPR-Cas12a-based protocol for the detection of SARS-CoV-2 with minimum equipment .…”

Microfluidic-based colorimetric nucleic acid detection of pathogens

“…20 In both cases, colorimetric (and fluorometric) changes can be assessed by the naked eye or with simple and inexpensive brightfield or fluorescence analysis setups, making colorimetric platforms inexpensive, portable, fast, and highly suitable for POC and low-resource settings. 21,22 In recent years and with the advent of the COVID-19 pandemic, the need for economic, sensitive, and portable nucleic acid detection platforms is in the spotlight. Colorimetric-readout platforms offer flexibility in output analysis and give a simplistic readout, which are especially crucial for resource-limited settings.…”

“…20 In both cases, colorimetric (and fluorometric) changes can be assessed by the naked eye or with simple and inexpensive brightfield or fluorescence analysis setups, making colorimetric platforms inexpensive, portable, fast, and highly suitable for POC and low-resource settings. 21,22…”

Nanozyme-catalyzed cascade reaction enables a highly sensitive detection of live bacteria

“…So far, various strategies have been proposed for accurate bacterial detections. [3][4][5][6][7][8][9][10][11] The most commonly used techniques are plate-counting methods, 7 enzyme-linked immunosorbent assays (ELISAs), 8 and polymerase chain reactions (PCRs). 9 In addition, electrochemical 10 and microfluidic 11 platforms are also frequently used nowadays.…”