Abstract
Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.
Highlights
It is important to accurately detect pathogens in clinical samples at very low concentrations [1,2]
Immunomagnetic separation (IMS) [5,6] and magnetic silica bead (MSB)-based DNA purification [7,8,9] are the most popular. These two technologies usually rely on handling small tubes containing several millilitres of the sample and manual operation; there is an urgent need for automated systems that can process large volumes of the samples simultaneously because higher concentrations of purified DNA can be obtained by preconcentrating the pathogens and purifying DNA from larger volumes of samples
Ten millilitres (10 mL) of phosphate-buffered saline (PBS) containing E. coli O157:H7 at 104 colony-forming units (CFU)/mL were mixed with 200 μL of MNPs (1013 particles/mL, final concentration) conjugated with affinity-purified E. coli O157 antibodies and the mixture was incubated at 37 ◦C and 200 rpm for 20 min in a beaker
Summary
It is important to accurately detect pathogens in clinical samples at very low concentrations [1,2]. When detecting pathogens in clinical samples, there are limitations because of the presence of substances that inhibit PCR [3,4] Such methods of detection still require sample pretreatment to isolate the target microorganisms and purify their nucleic acids [3,4,5]. Among these pretreatment techniques, immunomagnetic separation (IMS) [5,6] and magnetic silica bead (MSB)-based DNA purification [7,8,9] are the most popular. The performance of the device was verified by standard methods, such as colony counting, PCR, and quantitative PCR (qPCR)
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