Sunday, May 10, 2020

Three NSF RAPID Grants to Develop Quicker Test for COVID-19 for Holonyak Lab Faculty

Three Nick Holonyak Jr., Micro and Nanotechnology Lab (HMNTL) faculty members have received NSF Rapid Response Research (RAPID) program grants, all of which aim to shorten the amount of time it takes to process a COVID-19 test. Current tests can take as long as five days for results to be returned to the patient. Although more rapid nucleic acid tests that can give a result within an hour have become available, there are reports of a high rate of false negatives among these tests.

With the United States reaching the highest number of SARS-CoV-2 (this particular strain of coronavirus) cases out of any infected country, it is a national imperative to be able to test people before they show symptoms to reduce the spread of the deadly disease.

"As one of the only facilities in the country that incorporates both micro and nanofabrication cleanroom facilities and a BioNanotechnology Laboratory (BNL) under the same roof, HMNTL is proud to meet the moment and provide support to COVID-19 related essential research," said Xiuling Li, HMNTL interim director and Donald Biggar Willett Professor in Engineering.

Here is a more in-depth look at how HMNTL faculty are helping:

Rapid Electrical Detection of COVID-19 at Point-of-Care

A team led by Rashid Bashir, Dean of the Grainger College of Engineering, and Holonyak Lab faculty researcher, has proposed the development of a point-of-care device that uses nasal fluid samples to detect the presence of COVID-19 within 10 minutes.
Current tests are complex and labor-intensive, requiring each sample to be sent to a laboratory for confirmation. The test being developed by Bashir's group will simplify the process by eliminating the need to extract RNA from samples and simplify the test it as a whole. The new test will electrically detect specific nucleic acid molecules associated with the SARS-CoV2-2.

"Our approach can provide for a rapid electrical detection of the RNA amplification using graphene sensors and result in a miniaturized format for the test and also reduce the test's total processing time," said Bashir, Abel Bliss Professor of Engineering, professor of bioengineering, and member of the Center for Genomic Diagnostics.

The team hopes the proposed approach can be expanded beyond COVID-19 detection to become a global health technology that contributes to providing low-cost diagnostics of a number of viruses around the world in a portable and inexpensive way.

Rapid Single-Step Reagentless SARS-CoV-2 Viral Load Test by Detection of Intact Virus Particles

The next COVID-19 detection project combines capturing intact COVID-19 viruses with custom-designed DNA nanostructures so they can be immediately counted with a newly-invented type of biosensor imaging. This process could be completed and produce results in less than 15 minutes.

This new method would allow diagnostic facilities at the point of care to count each virus directly using a new form of ultrasensitive biosensor microscopy that amplifies the magnitude of light scattering produced by the virus when it is illuminated with a laser. To determine if the viewed virus is SARS-CoV-2, customized DNA nanostructure-based capture probes would be immobilized on a photonic crystal biosensor surface. When exposed to a sample, such as material eluted from a nasal swab, the DNA rhombus-shaped "virus net" would selectively attach the virus to the biosensor surface, while allowing all other materials to pass over the sensor without capture.

"Our approach would represent a new paradigm for virus diagnostics that does not require the chemical enzymatic amplification of nucleic acids, and so does not require temperature control, thermal cycles, viral lysis, nucleic acid purification, or fluorescent dyes," said Cunningham, Donald Biggar Willett Professor in Engineering and professor of electrical and computer engineering. "We just capture and count, so it is the simplest possible process, and our sensing method gives a result immediately as the viruses are captured."

The technology used in this method was recently demonstrated as a new form of biosensor microscopy called Photonic Resonator Interference Scattering Microscopy (PRISM), which allows researchers to detect and digitally count virus particles, protein molecules, and a variety of nanoparticles in real time without the use of additional labels or stains.

The team for this research also includes Xing Wang, associate professor of Chemistry, Taylor Canady, postdoc fellow at the Carl R. Woese Institute for Genomic Biology, and Nantao Li, Cunningham's ECE graduate student.

RAPID: Developing a Novel Biosensor for Rapid, Direct, and Selective Detection of COVID-19 using DNA Aptamer-Nanopore 

Holonyak Lab affiliate faculty member Yi Lu is working with Lijun Rong from the University of Illinois at Chicago to develop a biosensor that could detect and differentiate infectious SARS-CoV-2 from the SARS-CoV-2 that have been rendered noninfectious by patient's antibodies or disinfectants. This would allow patients to receive proper treatment in a timely manner, and would allow people who aren't infected or contagious to be released from quarantine.

The project aims to develop a modular and scalable sensor for direct detection of the intact coronavirus using DNA aptamers, short, single-stranded DNA molecules that can selectively bind infectious SARS-CoV-2. When coupled with nanopore, a pore of nanometer size, the result would be able to differentiate the infectious SARS-CoV-2 from the non-infectious forms or other viruses such as flu viruses with a high level of specificity.

"Achieving such a high level of specificity is very important for COVID-19 diagnostics," said Yi Lu, professor of chemistry and bioengineering. "This is because studies have shown that viral RNA levels that are being used in most COVID-19 diagnostic tests do not always correlate with viral transmissibility."

This technique is less resource-intensive than current methods due to not requiring pretreatment or RNA amplification. It also decreases the likelihood of cross contamination. It could also be used to test surface areas to ensure they have been properly sanitized after coming in contact with an infected patient.

COVID-19 Antigen Test Evaluated by European Scientists

Scientists in Europe recently evaluated the frontline capabilities of a commercially available, 15-minute disposable antigen test to detect COVID-19 infections.

Their findings, reported in Frontiers in Medicine, suggest the test could be useful as part of a broader triage strategy for slowing down the virus, which has infected more than seven million people and caused about 250,000 deaths as of May 4.

“The detection of viral infections in patients attending primary care centres would allow healthcare workers to rapidly identify new outbreak foci and define quarantine measures for high viral shedders and/or suspect patients to limit the spread of the epidemic,” the authors wrote.

The two-phase study examined the sensitivity and specificity of the new test during its development stage in the lab and later on using real-world biological samples from more than 300 previously infected patients.

Overall accuracy was 82 percent in the latter setting, with an overall sensitivity (how often a test correctly generates a positive result) of 57.6 percent and an overall specificity (how often a test correctly generates a negative result) of 99.5 percent.

In other words, the test was able to detect COVID-19 infections in about six out of 10 people, and it was nearly perfect in determining when an infection was not present. The test was more sensitive in patients with higher viral loads, positively identifying an infection in about seven out of 10 people.

The authors say the test — quicker, cheaper and less complicated but not as sensitive as reverse transcription-polymerase chain reaction (RT-PCR) assays, which ID the virus based on its genetic material — could be used to help screen patients during peak periods of the pandemic. Eventually, it could also be especially useful in screening higher-risk populations such as healthcare workers, they said.

The COVID-19 Ag Respi-Strip® test was developed by Belgian company Coris BioConcept, which specializes in rapid diagnostic kits for detecting respiratory and gastrointestinal pathogens like viruses and bacteria.

The test from Coris BioConcept is a type of immunochromatographic assay, or lateral flow test, which detects the presence or absence of a particular substance. Most people may be familiar with another type of lateral flow assay — a pregnancy test.

In the case of the COVID-19 Ag Respi-Strip, the antigen test uses a sample from a nasopharyngeal swab, which looks like a long, flexible Q-tip that enters through one nostril and extends down the nasal passage close to a person’s outer ear.

An antigen test works by looking for proteins on the surface of the virus. Coris BioConcept partly based the test on previous virology research on SARS-CoV-1, which caused the 2002-03 SARS epidemic. In fact, the two are so similar that the COVID-19 Ag Respi-Strip cannot differentiate between SARS-CoV-1 and -2.

The authors estimate the 15-minute antigen test, which can be conducted at point-of-care facilities following a few user-friendly protocols, could reduce the number of laboratory tests using RT-PCR by more than 13 percent.

Quidel Receives FDA Emergency Use Authorization for Rapid Antigen COVID-19 Diagnostic Assay

Quidel Corporation, a provider of rapid diagnostic testing solutions, cellular-based virology assays and molecular diagnostic systems, announced today that Quidel has received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA) to market its Sofia® 2 SARS Antigen FIA, a rapid point-of-care test to be used with the Sofia 2 Fluorescent Immunoassay Analyzer for the rapid detection of SARS-CoV-2 in nasal or nasopharyngeal specimens from patients meeting the Centers for Disease Control and Prevention’s (CDC) criteria for suspected COVID-19 infection.

Sofia 2 is Quidel’s next-generation version of its best-selling Sofia instrumented system. Sofia 2 utilizes the original Sofia fluorescent chemistry design while improving upon the graphical user interface and optics system to provide an accurate, objective and automated result in 15 minutes. The next-generation Sofia 2 system also comes connected to Virena®, Quidel’s data management system, which provides aggregated, de-identified testing data in near real-time.

The Sofia 2 instrument also offers 2 distinct workflows: depending upon the user’s choice, the Sofia 2 SARS Antigen FIA cartridge is placed inside Sofia 2 for automatically timed development (WALK AWAY Mode); or test cartridges can be placed on the counter or bench top for a manually timed development and then placed into Sofia 2 to be scanned (READ NOW Mode), allowing the user to markedly increase testing throughput per hour.

“In the fight against COVID-19, our employees are truly making a difference, and I am tremendously proud of our organization’s ability to quickly develop and mobilize an accurate rapid antigen test,” said Douglas Bryant, president and chief executive officer of Quidel Corporation. “The EUA for our Sofia 2 SARS Antigen FIA allows us to arm our healthcare workers and first responders with a frontline solution for COVID-19 diagnosis, accelerating the time to diagnosis and potential treatment of COVID-19 for the patient.”

The assay is currently available for sale in the United States under EUA, and Quidel is now shipping the product to its customers. Quidel offers several other Sofia assays for sale, which are FDA cleared and CLIA waived, including tests for Influenza A and B, Respiratory Syncytial Virus (RSV), Group A Strep, and a 12-minute finger-stick whole blood test for Lyme Disease. In addition, Quidel also markets Sofia® tests for Lyme Disease, Legionella and S. pneumoniae in Europe.

Friday, May 01, 2020

CRISPR-based Technology Spots COVID-19

Researchers have developed a new technology that flexibly scales up CRISPR-based molecular diagnostics, using microfluidics chips that can run thousands of tests simultaneously. A single chip’s capacity ranges from detecting a single type of virus in more than 1,000 samples at a time to searching a small number of samples for more than 160 different viruses, including the COVID-19 virus.

Called Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), this technology — validated on patient samples — provides same-day results and could someday be harnessed for broad public-health efforts.

The work appears in Nature, led by co-first authors Cheri Ackerman and Cameron Myhrvold, both postdoctoral fellows at the Broad Institute of MIT and Harvard. Paul Blainey, core member of the Broad Institute and associate professor in the Department of Biological Engineering at MIT, and Pardis Sabeti, institute member at Broad, professor at Harvard University, and Howard Hughes Medical Institute Investigator, are co-senior authors.

“The current pandemic has only underscored that rapid and sensitive tools are critical for diagnosing, surveilling, and characterizing an infection within a population,” said Sabeti. “The need for innovative diagnostics that can be applied broadly in communities has never been more urgent.”

“CRISPR-based diagnostics are an attractive tool for their programmability, sensitivity, and ease of use,” said Myhrvold. “Now, with a way to scale up these diagnostics, we can explore their potential for comprehensive approaches — for example, enabling clinicians to see if patients are harboring multiple infections, to rule out a whole panel of diseases very quickly, or to test a large population of patients for a serious infection.”

Miniaturizing CRISPR diagnostics

To build a testing platform with this capacity, the team turned to microfluidics, adapting and improving on technology developed in 2018 by Blainey’s lab. The researchers created rubber chips, slightly larger than a smartphone, with tens of thousands of “microwells” — small compartments designed to each hold a pair of nanoliter-sized droplets. One droplet contains viral genetic material from a sample, and the other contains virus-detection reagents.

“The microwell chips are made like a stamp — it’s rubber poured over a mold,” explained Ackerman. “We’re easily able to replicate and share this technology with collaborators.”

The detection approach used on the chips is adapted from the CRISPR-based diagnostic SHERLOCK, first described in 2017 and developed by team of scientists from the Broad Institute, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering & Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University.

To use the CARMEN platform, researchers first extract viral RNA from samples and make copies of this genetic material, similar to the preparation process for RT-qPCR diagnostics currently used for suspected COVID-19 cases. The researchers then add a unique fluorescent color dye to each prepared sample and divide the mixture into tiny droplets.

The detection mixtures, on the other hand, contain the CRISPR protein Cas13, a guide RNA that looks for a specific viral sequence, and molecules to report the results. These mixtures are also color-coded and separated into droplets.

Thousands of droplets from the samples and detection mixtures are then pooled together and loaded onto a chip in a single pipetting step. Each microwell in the chip catches two droplets. When a detection droplet finds its target — a specific viral genetic sequence — in a sample droplet in the same microwell, a signal is produced and detected by a fluorescence microscope. The entire protocol, from RNA extraction to results, takes under eight hours.

“Uniting these two technologies in a single platform gives us exciting new capabilities to investigate clinical and epidemiological questions,” said co-author Gowtham Thakku, an MIT graduate student in Broad’s Infectious Disease and Microbiome Program.

CARMEN enables more than 4,500 tests on a single microfluidics chip, which can apply to patient samples in a variety of ways using the available fluorescent codes. For example, a single chip could simultaneously test 1,048 samples for a single virus, or five samples for 169 viruses. The capacity can be easily scaled up further by adding more chips: “We normally run four or five chips in a single day,” noted Ackerman.

Multiplexing capabilities

To showcase the platform’s multidiagnostic capabilities, the team developed a strategy for rapidly testing dozens of samples for the 169 human-associated viruses that have more than 10 published genome sequences. The researchers tested this detection panel against 58 patient samples, using multiple chips. They additionally applied CARMEN on patient samples to differentiate between subtypes of influenza A strains and to detect drug-resistance mutations in HIV.

The team also incorporated detection mixtures for SARS-CoV-2 — the virus that causes COVID-19 — and other respiratory pathogens to demonstrate, using synthetic viral sequences, how the assay can be rapidly adapted to detect emerging viruses.

“CARMEN offers both impressive throughput and flexibility in diagnostic testing,” said co-author Catherine Freije, a Harvard graduate student in the Sabeti lab.

The researchers report that the platform’s sensitivity is comparable to previously published SHERLOCK assays, and they are continuing to improve and validate CARMEN using additional clinical samples. Coupled with the successful testing data from patient samples described in Nature today, this approach could be readily translatable in the clinic, according to the team.

“This miniaturized approach to diagnostics is resource-efficient and easy to implement,” said Blainey. “New tools require creativity and innovation, and with these advances in chemistry and microfluidics, we’re enthusiastic about the potential for CARMEN as the community works to beat back both COVID-19 and future infectious disease threats.”

Support for this study was provided in part by Howard Hughes Medical Institute, the Koch Institute for Integrative Cancer Research Bridge Project, an MIT Deshpande Center Innovation Award, the Merkin Institute for Transformative Technologies in Healthcare, a Burroughs Wellcome Fund CASI Award, the Defense Advanced Research Projects Agency (DARPA) grant D18AC00006, and the NIH (F32CA236425).

Source: The Havard Gazette

Mammoth Biosciences Announces Peer-Reviewed Validation Of Its Rapid, CRISPR-Based COVID-19 Diagnostic

Mammoth Biosciences announced the publication of a study demonstrating the power of its platform to detect SARS-CoV-2 from respiratory swab RNA extracts in under 45 minutes. The study, published in Nature Biotechnology, contains the first peer-reviewed data using CRISPR diagnostics for COVID-19, with the largest set of patient samples to-date.

There is an urgent need for rapid and accessible testing of the novel coronavirus for an effective public health response. The current and most common method for diagnosing SARS-CoV-2 is through quantitative polymerase chain reaction (qRT-PCR), which is restricted for use within specialized laboratories. As a result, the typical turnaround time for screening and diagnosing patients with suspected SARS-CoV-2 has been more than 24 hours, a pace that is far too slow to keep up with such a contagious disease.

Mammoth Biosciences has harnessed the power of CRISPR to offer a faster, lower-cost and visual alternative to traditional qRT-PCR assays. The company’s CRISPR-based diagnostic assay, DETECTR™, can deliver results in under 45 minutes as visualized on a lateral flow strip, similar to an at-home pregnancy test. DETECTR does not require a complex laboratory setting; it can be performed with portable heat blocks and readily available, “off-the-shelf” reagents and disposable lateral flow strips. The assay offers similar levels of sensitivity and specificity to qRT-PCR tests, with 95 percent positive predictive agreement and 100 percent negative predictive agreement.

The study was led by infectious disease expert Dr. Charles Chiu, researchers from the Department of Laboratory Medicine at the University of California, San Francisco (UCSF), along with Mammoth’s Chief Technology Officer Dr. Janice Chen and Research Lead Dr. James Broughton, and the California Department of Public Health. The researchers validated the method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections.

“We need faster, more accessible and scalable diagnostics. The point-of-care testing space is ripe for disruption and CRISPR diagnostics have the potential to bring reliable testing to the most vulnerable environments.,” says Mammoth’s Chief Technology Officer Janice Chen. “Because CRISPR can be programmed to detect any DNA or RNA sequence, we have been able to reconfigure our DETECTR platform within days to detect the SARS-CoV-2 virus from one of the first confirmed cases in the U.S., made possible by our collaboration with Dr. Charles Chiu at UCSF.”

From its inception, Mammoth Biosciences has focused on leveraging the diagnostic capabilities of CRISPR to develop decentralized, point-of-care tests for a variety of diseases. Shortly after the outbreak of SARS-CoV-2 in Wuhan, China, Mammoth began building its COVID-19 diagnostic and validated the efficacy of its CRISPR-based protocols on patient samples in less than two weeks. In mid-February, Mammoth published its white paper, open-sourcing its CRISPR-based detection protocols, and in early March, published the preprint containing initial patient sample validation.

Monday, April 27, 2020

Stream Bio and MIP Diagnostics Working on COVID-19 Rapid Diagnostic and Mass Screening Test

Stream Bio, a company that develops and manufactures a range of bioimaging molecular probes, has announced a new joint venture with MIP Diagnostics Ltd., a company that develops molecular imprinting for diagnostic and other applications. The collaborative project will focus on the development of a COVID-19 (or SARS-Cov-2) antigen reagent for assays, a lateral flow Rapid Diagnostic Test (RDT) and an “ELISA” type assay for high throughput screening (HTS) or mass testing.  

The unique properties of both Stream’s and MIP’s novel technologies are intended to allow for fast development of an extremely sensitive, and stable detection platform for the virus. The lateral flow project aims to reduce the diagnosis time to just 10 minutes, while the ELISA assay would enable a different detection system common in nearly all labs to be utilized alongside PCR, dramatically increasing capability.

With current PCR-based methods, it can take over a day to receive and act on lab results. The proposed point-of-care lateral flow technology (LFT) could reduce this by more than 140 times and once validated, be deployed anywhere for “on-the-spot” screening, for use by first responders on scene to transit hubs and airports.  The resulting LFT strip can easily be mass-produced.  

In this consortium, Stream Bio’s Conjugated Polymer Nanoparticles (CPNs) will combine their capabilities for temperature stability, intense fluorescence and magnetism, with the versatile, stable molecular imprinted polymers (nanoMIPs) or synthetic “plastic antibodies” from MIP Diagnostics Ltd.. The proprietary nanoMiPs work in the same way as conventional antibodies by targeting and latching onto a specific “binding site” of the virus, the “spike”, but without the significant development timeline or immunogenic requirement.  

Andy Chaloner, Founding Director and CEO of Stream Bio, commented “I am extremely excited by the possibilities of the combination of our two technologies, and the novel angle we can bring to the fight against the COVID-19 pandemic. CPNs have previously shown great capabilities in diagnostics, and implementing them in this collaboration is a huge milestone for Stream Bio.”  

“This is another great opportunity for nanoMIPs to make a significant impact on diagnostics development by leveraging the fast turnaround, high robustness and sensitivity benefits of MIPs with CPNs” said Stephane Argivier, Interim CEO, MIP Diagnostics Ltd., “and we are pleased to be working with another innovative platform on this collaboration to make a significant impact on the current worldwide need for rapid test development to COVID-19 diagnosis and monitoring.”

Portable Microfluidic Platform Developed for Detecting Coronavirus Using Smartphone

Researchers headed by a team at the University of Illinois, Urbana-Champaign, have developed what they claim is an inexpensive, sensitive smartphone-based device that can detect viral and bacterial pathogens in about 30 minutes, and could be adapted to test for SARS-CoV-2. The platform comprises a cartridge-housed microfluidic chip that carries out isothermal amplification of viral nucleic acids from nasal swab samples, which are then detected using the smartphone camera. The investigators report on their use of the system to detect equine viruses as a non-biohazard surrogate for SARS-CoV-2, but say that when adapted to test for coronavirus, the smartphone accessory, costing about $50, could be used to reduce the pressure on testing laboratories during pandemics such as COVID-19.

“This test can be performed rapidly on passengers before getting on a flight, on people going to a theme park, or before events like a conference or concert,” said University of Illinois, Urbana-Champaign electrical and computer engineering professor Brian Cunningham, PhD, who, together with bioengineering professor Rashid Bashir, PhD, led the development of the device. “Cloud computing via a smartphone application could allow a negative test result to be registered with event organizers or as part of a boarding pass for a flight. Or, a person in quarantine could give themselves daily tests, register the results with a doctor, and then know when it’s safe to come out and rejoin society.”

The multi-institutional researchers described their development and use of the device, in Lab on a Chip. The paper is titled, “Smartphone-Based Multiplex 30-minute Nucleic Acid Test of Live Virus from Nasal Swab Extract.”

As the COVID-19 pandemic has escalated, a “key failure” of health systems across every country has been the ability to rapidly and accurately diagnose disease, the authors stated. Contributing factors include “ … a limited number of available test kits, a limited number of certified testing facilities, combined with the length of time required to obtain a result and provide information to the patient.”

Most viral test kits rely on labor- and time-intensive laboratory preparation and analysis techniques, they continued. Testing for SARS-CoV-2 from nose swabs can take days. And “because available technologies remain expensive (in terms of capital equipment and reagents), technically challenging, and labor intensive, there is an urgent need for low-cost portable platforms that can provide fast, accurate, and multiplex diagnosis of infectious disease at the point of care,” the researchers pointed out. “The challenges associated with rapid pathogen testing contribute to a lot of uncertainty regarding which individuals are quarantined and a whole host of other health and economic issues,” Cunningham said.

Nucleic acid tests (NATs) represent an important class of point-of-care (POC) technologies for pathogen sensing that can achieve high specificity for the detection of pathogenic nucleic acid sequences, the authors noted. Such tests can also be designed to tag the amplified sequences using fluorometric or colorimetric markers. “Due to their success in laboratory settings, considerable efforts have been devoted to performing NATs in POC settings,” the investigators commented. While most NAT methods are based on polymerase chain reaction (PCR) amplification, which requires repeated heating and cooling cycles that are not ideal for POC applications, NATs that use isothermal nucleic acid amplification approaches, such as loop-mediated isothermal amplification (LAMP) are now being harnessed to develop simple, miniaturized POC devices. LAMP can rapidly amplify nucleic acids at a constant temperature, with just one type of enzyme and four to six primers.


The device developed by Cunningham and Bashir’s team started out as a project to detect a panel of equine viral and bacterial pathogens, including those that cause severe respiratory illnesses in horses, which are similar to those presented in COVID-19. “Utilizing the system in the context of equine respiratory diseases represents a model system for human pathogens such as SARS-CoV-2, which does not pose biosafety issues, but preserves the main features of a human COVID-19 testing protocol,” the researchers indicated. “Horse pathogens can lead to devastating diseases in animal populations, of course, but one reason we work with them has to do with safety,” Cunningham noted.

“The horse pathogens in our study are harmless to humans.” The researchers developed a device that could detect multiple horse viral pathogens quickly and cost-effectively, using LAMP technology. The device comprises a small cartridge containing the testing reagents and a port to insert a nasal extract or blood sample. The whole unit then clips to a smartphone. The test reagents break open the viral pathogens to gain access to the RNA. A primer molecule then amplifies the genetic material into many millions of copies in about 10 or 15 minutes. A fluorescent dye then stains the copies and glows green when illuminated by blue LED light, which is detected by the smartphone’s camera.

Recent research has consistently shown that image sensors integrated within today’s smartphones have enough sensitivity to detect fluorescence in the contexts of fluorescence microscopy of cells, viruses, and bacteria. Smartphone cameras can also sense fluorescence signals from a wide variety of biological assays, including LAMP, within microfluidic compartments, the authors noted. “The advantage of using a smartphone as the detection instrument for POC analysis is that it is possible to take advantage of the integrated optics, image sensor, computation power, user interface, and wireless communication capabilities of mobile devices, thus minimizing cost,” the team wrote.” With assistance from an inexpensive snap-in cradle or clip-on instrument, anyone that carries a smartphone would have the ability to perform testing.”

The investigators used their prototype device to detect nucleic acids from five different pathogens that cause equine respiratory infectious diseases. “Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ~30 minutes,” they wrote. And while the test system reported in the paper was used to detect pathogenic DNAs, the assay could be easily adapted for detecting RNA viruses, they suggested, by using a one-step RT-LAMP protocol that adds reverse transcriptase to the LAMP reaction mix without modifying the buffer or reaction conditions.

They suggest that using a smartphone in conjunction with a cradle that enables the phone’s camera to quickly gather a fluorescent endpoint image of the LAMP reaction, it will be possible to generate a positive/negative result, and incorporate integrated experimental controls and replicates to assure that the test has been carried out correctly. “ … we envision a detection instrument that clips onto a smartphone, with mechanical adapters that will align the rear-facing camera correctly with several popular phone models,” the scientists explained.

Using a mobile device as a detection instrument will also make it possible to report the data via integration with telemedicine platforms, both for epidemiology reporting and for sharing test results with doctors.

The researchers’ system does currently require a few preparatory steps to be performed outside of the device, but they are working on a cartridge that has all of the reagents needed to create a fully integrated system. “In future work, our plans include integrating the functions of viral lysis, LAMP buffer mixing, and LAMP reaction into a single cartridge with the reagents held within on-cartridge reservoirs,” they wrote.

Other researchers at the University of Illinois are using the novel coronavirus genome to create a mobile test for COVID-19, and making an easily manufactured cartridge that Cunningham said would improve testing efforts.

Source: Genetic Engineering & Biotechnology News

Monday, April 20, 2020

McKelvey Engineering Researchers Receives Funding for Rapid COVID-19 Test Based on Ultrabright Fluorescent Nanoprobe Technology

Engineers at the McKelvey School of Engineering at Washington University in St. Louis have received federal funding for a rapid COVID-19 test using a newly developed technology.

Srikanth Singamaneni, professor of mechanical engineering and materials science, and his team have developed a rapid, highly sensitive and accurate biosensor based on an ultrabright fluorescent nanoprobe, which has the potential to be broadly deployed.

Called plasmonic-fluor, the ultrabright fluorescent nanoprobe can also help in resource-limited conditions because it requires fewer complex instruments to read the results.

Singamaneni hypothesizes their plasmonic-fluor-based biosensor will be 100 times more sensitive compared with the conventional SARS-CoV-2 antibody detection method. Increased sensitivity would allow clinicians and researchers to more easily find positive cases and lessen the chance of false negatives.

Plasmonic-fluor works by increasing the fluorescence signal to background noise. Imagine trying to catch fireflies outside on a sunny day. You might net one or two, but against the glare of the sun, those little buggers are difficult to see. What if those fireflies had the similar brightness as a high-powered flashlight?

Plasmonic-fluor effectively turns up the brightness of fluorescent labels used in a variety of biosensing and bioimaging methods. In addition to COVID-19 testing, it could potentially be used to diagnose, for instance, that a person has had a heart attack by measuring the levels of relevant molecules in blood or urine samples.

Using plasmonic-fluor, which is composed of gold nanoparticles coated with conventional dyes, researchers have been able to achieve up to a 6,700-fold brighter fluorescent nanolabel compared with conventional dyes, which can potentially lead to early diagnosis. Using this nanolabel as an ultrabright flashlight, they have demonstrated the detection of extremely small amounts of target biomolecules in biofluids and even molecules present on the cells.

The study was published in the April 20 issue of Nature Biomedical Engineering.

Gold nanoparticles serve as beacons

In biomedical research and clinical labs, fluorescence is used as a beacon to see and follow target biomolecules with precision. It’s an extremely useful tool, but it’s not perfect.

“The problem in fluorescence is, in a lot of cases, it’s not sufficiently intense,” Singamaneni said. If the fluorescent signal isn’t strong enough to stand out against background signals, just like fireflies against the glare of the sun, researchers may miss seeing something less abundant but important.

“Increasing the brightness of a nanolabel is extremely challenging,” said Jingyi Luan, lead author of the paper. But here, it’s the gold nanoparticle sitting at the center of the plasmonic-fluor that really does the work of efficiently turning the fireflies into flashlights, so to speak. The gold nanoparticle acts as an antenna, strongly absorbing and scattering light. That highly concentrated light is funneled into the fluorophore placed around the nanoparticle. In addition to concentering the light, the nanoparticles speed up the emission rate of the fluorophores. Taken together, these two effects increase the fluorescence emission.

Essentially, each fluorophore becomes a more efficient beacon, and the 200 fluorophores sitting around the nanoparticle emit a signal that is equal to 6,700 fluorophores.

In addition to detecting low quantities of molecules, sensing time can be shortened using plasmonic-fluor as brighter beacons mean fewer captured proteins are needed to determine their presence.

The researchers have also shown that plasmonic-fluor allows the detection of multiple proteins simultaneously. And in flow cytometry, plasmonic-fluor’s brightening effect allows for a more precise and sensitive measurement of proteins on cell surface, whose signal may have been buried in the background noise using traditional fluorescent tagging.

There have been other efforts to enhance fluorescent tagging in imaging, but many require the use of an entirely new workflow and measurement platform. In addition to plasmonic-fluor’s ability to greatly increase the sensitivity and decrease the sensing time, it doesn’t require any changes to existing laboratory tools or techniques.

The technology has been licensed to Auragent Bioscience LLC by Washington University’s Office of Technology Management. Auragent is in the process of further development and scaling up the production of plasmonic-fluors for commercialization.

This work was supported by the National Science Foundation (CBET-1512043 and CBET 1254399); the National Institutes of Health (R01 DE02709802, R01 CA141521 and U54 CA199092); and a grant from the Barnes-Jewish Hospital Research Foundation (3706).

RIT Researchers Build Lab-on-a-Chip and Magnetic Nano-bead Device to Detect Bacteria and Viruses

Engineering researchers developed a next-generation miniature lab device that uses magnetic nano-beads to isolate minute bacterial particles that cause diseases. Using this new technology improves how clinicians isolate drug-resistant strains of bacterial infections and difficult-to-detect micro-particles such as those making up Ebola and coronaviruses.

Ke Du and Blanca Lapizco-Encinas, both faculty-researchers in Rochester Institute of Technology’s Kate Gleason College of Engineering, worked with an international team to collaborate on the design of the new system—a microfluidic device, essentially a lab-on-a-chip.

Drug-resistant bacterial infections are causing hundreds of thousands of deaths around the world every year, and this number is continuously increasing. Based on a report from the United Nations, the deaths caused by antibiotics resistance could reach to 10 million annually by 2050, Du explained.

“It is urgent for us to better detect, understand, and treat these diseases. To provide rapid and accurate detection, the sample purification and preparation is critical and essential, that is what we are trying to contribute. We are proposing to use this novel device for virus isolation and detection such as the coronavirus and Ebola,” said Du, an assistant professor of mechanical engineering whose background is in development of novel biosensors and gene editing technology.

The lab team is interested in the detection of bacterial infection, especially in bodily fluids. One of the major problems for detection is how to better isolate higher concentrations of pathogens.

The device is a sophisticated lab environment that can be used in field hospitals or clinics and should be much faster at collecting and analyzing specimens than the commercially available membrane filters. Its wide, shallow channels trap small bacteria molecules that are attracted to packed, magnetic microparticles.

This combination of the deeper channels on the nano-device, increased flow rate of fluids where bacteria are suspended, and the inclusion of magnetic beads along the device channels improves upon the process of capturing/isolating bacterial samples. Researchers were able to successfully isolate bacteria from various fluids with a microparticle-based matrix filter. The filter trapped particles in small voids in the device, providing a larger concentration of bacteria for analysis. An added advantage of a smaller device such as this allows for multiple samples to be tested at the same time.

“We can bring this portable device to a lake which has been contaminated by E. coli. We will be able to take a few milliliters of the water sample and run it through our device so the bacteria can be trapped and concentrated. We can either quickly detect these bacteria in the device or release them into certain chemicals to analyze them,” said Du, whose earlier work focused on devices that use the CRISPR gene-editing technology and the fundamental understanding of fluidic dynamics.

Teaming up with Lapizco-Encinas, a biomedical engineer with expertise in dielectrophoresis—a process that uses electrical current to separate biomolecules—their collaboration provided the increased capability toward better pathogen detection, specifically for bacteria and microalgae isolation and concentration.

“Our goal is not only isolating and detecting bacteria in water and human plasma, but also working with whole blood samples to understand and detect blood infection such as sepsis. We already have a concrete plan for that. The idea is to use a pair of the nano-sieve devices for sequential isolation,” said Lapizco-Encinas, an associate professor in RIT’s biomedical engineering department.

Du and Lapizco-Encinas were part of a team that consisted of mechanical and biomedical engineers from Rutgers, University of Alabama, SUNY Binghamton, and Tsinghua-Berkeley Shenzhen Institute in China to address the global challenges of disease pandemics. The new data is published in the article “Rapid Escherichia coli trapping and retrieval for bodily fluids via a three-dimensional bead-stacked nano-device,” in the journal ACS Applied Materials and Interfaces.

The research team is RIT engineering doctoral and graduate students Xinye Chen, Abbi Miller and Qian He; University of Alabama assistant professor of electrical and computer engineering Yu Gan and undergraduate student Shengting Cao; Ruo-Qian Wang, assistant professor of civil and environmental engineering from Rutgers University; Xin Yong, assistant professor of mechanical engineering from SUNY Binghamton; Peiwu Qin from the Center of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute, China; and Jie Zhang, Carollo Engineers Inc. in Seattle.