NEW YORK – A team of researchers affiliated with the Massachusetts Institute of Technology's research enterprise in Singapore is developing a novel sample preparation method aimed at enriching samples for low-abundance microorganisms.
Led by Jongyoon Han, a principal investigator at Singapore-MIT Alliance for Research and Technology's (SMART) department of Critical Analytics for Manufacturing Personalized-Medicine (CAMP), the team has shown the method can be used in conjunction with different nucleic acid amplification-based diagnostics.
The method uses electrostatic microfiltration, Han said in an interview, and the team now hopes to partner with industry to develop further applications.
In a quirk of biology that has been known for more than 100 years, peptidoglycan on the outer surface of bacterial cells gives them a negative charge at neutral pH and causes them to dance toward the anode in an electric field.
The SMART enrichment technology exploits this quirk to capture negatively charged pathogens, ultimately concentrating microorganisms from 150- to 8,000-fold, Han said.
"The capture efficiency can be as high as 90 percent for an abundance as low as 10 colony forming units per milliliter," he said. The team has also shown that the enriched microorganisms can be detected through digital loop-mediated isothermal amplification (dLAMP) and sequencing platforms, he added.
The researchers paired the method with qualitative LAMP-based targeted detection and with nanopore sequencing-based non-targeted detection that can indetify microorganism species and potentially antimicrobial resistance.
"The current results demonstrated a 100x improvement in the sensitivity, or limit of detection, for both LAMP and sequencing-based detection," he said.
Han's team worked with the SMART Antimicrobial Resistance interdisciplinary research group on the project beginning in 2021. The work was recently awarded funding from the National Research Foundation of Singapore, and one of the team members, Yaoping Liu, was also awarded a seed grant from the same agency, Han said.
The team filed a US patent application for the technology in September, and has presented the microfiltration method at two recent conferences.
At the International Conference on Miniaturized Systems for Chemistry and Life Sciences in Hangzhou, China last year, the researchers showed proof-of-concept data.
At the European Congress of Clinical Microbiology & Infectious Diseases (ECCMID) in April, they presented a poster demonstrating that the method enriches low-abundance bacteria from large volumes of sample and can improve the performance of digital PCR and sequencing.
The team specifically showed that samples with approximately 104 CFU of Staphylococcus aureus bacteria in 1 ml processed at a throughput greater than 1 ml/minute had a capture efficiency of approximately 90 percent. The method enabled a detection improvement of approximately 40-fold compared to samples that were simply centrifuged to concentrate pathogens.
The team described the filter as being coated with a Ca-alginate gel to give it a positive charge and reduce the pore size to approximately 1.25 microns. They showed that the filter is inserted in a tube with the sample and spun in a centrifuge. The pathogens adhere to the filter, and the positively charged gel can then be removed with EDTA to release the live bacteria, which can subsequently be cultured.
They also paired the electrostatic microfiltration method with a recently published warm-start CRISPR/Cas-based digital LAMP technique, demonstrating improved bacterial capture efficiency from 20 percent to 90 percent compared to centrifugation alone. The method also increased the detected dLAMP signals from approximately 30 copies per microliter to more than 1,000 copies per microliter, according to the poster.Han said that the technology has "universal applicability" for pathogens from bacteria and fungi to viruses. It can also be used to detect pathogens in samples from diverse sources, such as wastewater, cell therapy products, and clinical specimens.
Importantly, the team has shown that the method has "good compatibility with various downstream detection platforms," such as PCR and sequencing, and that it is also portable, fast, and easy to use, Han said.
The microfilter used in the technique was invented during Liu's doctoral research at Peking University in China and Liu and colleagues described it in a 2018 Microsystems & Nanoengineering study. The technology has since been licensed to an undisclosed startup company for commercialization, Han said, and the microfilters used in developing the team's technique were purchased from that company.
"The cost of the microfilter is the major cost of goods for the system and is closely related to the manufacturing quantity," he added.
The researchers continue to improve the workflow's limit of detection by further optimizing the Ca-alginate coating thickness, filtration throughput, and release conditions.
"The team is more than happy to talk and collaborate with companies interested in commercializing this technique," he said, noting that the group is currently in discussions with a few undisclosed firms.