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Momentum Builds for Amplification-Free Molecular Diagnostics, but Significant Hurdles Remain


NEW YORK – Nucleic acid amplification technologies (NAATs) are both the bedrock and biggest bottleneck of molecular diagnostics, enabling sensitive and specific infectious disease assays that also tend to be complex and expensive.

A panoply of new approaches is now accelerating toward commercialization, including a few for amplification-free detection of pathogen DNA or RNA in point-of-care and home settings.

Amplification-free molecular approaches often mix and match exotic-sounding capture elements — like DNA origami, aptamers, gold nanodimples, or CRISPR — with cutting-edge detection technologies, such as quantum dots, graphene field effect transistors (FETs), surface plasmon resonance, and nanopores.

In the past few years, a number of startups have debuted with a proposition to use these component technologies to make rapid, inexpensive molecular diagnostic tests, accelerated by pandemic-era scientific and manufacturing innovations to support cheaper and more decentralized testing.

However, the "valley of death" of clinical trials and regulatory clearance remains a formidable obstacle to the development and adoption of these assays for clinical care. Although the core technologies are exciting, startups with amplification-free MDx tests may face an uphill climb with regulatory authorities, particularly the US Food and Drug Administration, since there are no obvious authorized predicate devices. They may also face competition from established players in point-of-care and at-home molecular diagnostics, some of whom are themselves struggling to find a niche.

Even discussing amplification-free molecular diagnostics as a group is a bit tricky, as they tend to be described in the broadest terms by what they are not, using terms like non-NAAT, non-PCR, or non-amplification.

Furthermore, some of these approaches can also detect other biomarkers, such as proteins, so it may not even be appropriate to pigeonhole them as molecular diagnostics.

Calling them "single-molecule" technologies can capture the potential for target variability, suggested Holger Schmidt, a professor of electrical engineering and optoelectronics at the University of California, Santa Cruz.

Binning these single-molecule, amplification-free approaches by their readout methods — electrical versus optical, for example — may also be an effective way to describe these newcomers en masse, he suggested.

Along with longtime collaborators at Brigham Young University and the University of Illinois Urbana-Champaign, Schmidt has developed an approach using microbead-based target capture, optical trapping, and nanopores to detect nucleic acids as well as proteins and other molecules. The approach was recently used to successfully track viral loads in primate body fluids in the lab.

Schmidt cofounded a startup called Fluxus in 2017 to help bring this technology to market. The company, now a subsidiary of Fujirebio, is still performing early development work to address manufacturability and market readiness, but how the tech may be incorporated into future commercial products remains to be determined.

In another bench-to-bedside project, bioengineer Brian Cunningham and his team at UIUC have been combining biosensor microscopy with nanoparticle tags such as quantum dots, gold nanoparticles, and plasmonic fluorophores.

The team uses photonic crystal surfaces to amplify detection, Cunningham said, such that individual tags are detectable with an inexpensive instrument incorporating a webcam-quality image sensor and an inexpensive light source, such as a red LED light. 

Cunningham's group has been using alternative molecular biology methods rather than PCR, including "nucleic acid strand displacement reactions that are extremely specific for their targets, not impacted by inhibitors, operate at room temperature, and don't require enzymes," he said.

The team is also developing approaches that leverage CRISPR-Cas in an amplification-free protocol, Cunningham said, as well as assays that recycle the target molecule to generate more nanoparticles for counting. In that sense, the approach is a form of amplification that is more linear than exponential, and thus is not inclined to runaway false amplification. The sample-to-answer method produces results in 10 minutes with a limit of detection of approximately 1 attomolar at room temperature, he said.

Cunningham also cofounded a startup, Atzeyo Biosensors, last year to commercialize some of these assays and instruments. 

Usurping NAATs?

Most of the known amplification-free startups are targeting the point-of-care and at-home markets. The home testing space seems to be exceptionally sensitive to cost, so, assuming amplification-free approaches are less expensive to produce than, for instance, isothermal amplification-based tests and instruments, they could have a leg up. 

The multitude of companies that launched point-of-care and handheld nucleic acid amplification systems and diagnostics for infectious diseases during the COVID pandemic have worked to expand and diversify test menus, but some are now being pruned.

"The pandemic has provided more grounding in which technologies can be developed into effective sample-to-result diagnostic tests, and which are still in earlier stages of development," said Sam Sia, a researcher at Columbia University with point-of-care testing expertise.

From his perspective, Sia is partial to PCR, and a company he cofounded, RoverDx, is developing all-optical point-of-care PCR.

"I think a challenge with amplification-free approaches is still a low limit of detection," he said.

Because of this, most approaches still require a pre-amplification step, he said. Though there are some that propose to bypass amplification, whether they can reach the promised limits of detection is still a work in progress, he added. "For now, PCR remains the gold standard, even after many challenges."

So, although it is sometimes touted as a replacement for PCR and isothermal amplification, a more tempered assessment of amplification-free MDx might be that certain use cases may benefit from these approaches.

Wilson Zhang, CEO of CRISPR-based diagnostics firm Intelligenome, said that none of the amplification-free technologies he is aware of seem more exciting than others or more likely to be commercially developed in the near term. However, two Alzheimer's disease therapies approved by the FDA have led to firms like Quanterix developing direct diagnostic tests using blood samples rather than cerebrospinal fluid, he said, and for this purpose, "single-molecule detection technology is fantastic," as it can detect down to femtogram-per-microliter levels.

"Amplification-free technologies may possibly replace PCR or isothermal amplification if the analytical and clinical data support it," Zhang said. However, "the technologies must work well for unmet needs in the market, and are accepted only when costs are low, turnaround time is quick, and the tests are easy to conduct."

That said, amplification-free methods usurping PCR is not entirely science fiction to UCSC's Schmidt.

"It's certainly possible, as more and more methods are reaching the sensitivity — as in limit of detection — of PCR or are at least able to detect all relevant concentrations while eliminating the additional processing steps," he said.


Innovations in device fabrication and miniaturization, assay chemistries like CRISPR, photonics, and nanopore-, graphene-, and FET-based electrical sensing mechanisms, are all driving progress in the field, Schmidt noted.

Many of the technologies underpinning amplification-free diagnostics are not terribly new, but the science seems to be accelerating. For example, a cursory search of the National Institutes of Health Reporter grants database for amplification-free diagnostics yields 148 funded projects in the past four-and-a-half years, 77 projects in the prior five years, and 51 in the five years between 2014 and 2010. Currently, 28 "amplification-free" NIH-funded projects are active. And, a quick PubMed search of the term amplification-free yields 43 results published in 2020, climbing to 107 in 2023.

Even some older approaches have seen exciting new developments of late. For example, researchers recently directly detected RNA of SARS-CoV-2 using a fluorogenic DNA aptamer, called lettuce, that activates a molecule resembling green fluorescent protein.

Similarly, platforms using the optical methods of plasmonic resonance or surface-enhanced Raman spectroscopy have been in development for decades, according to a recent review of their use for virus detection, but new approaches, such as using nanodimples with internalized gold nanoparticles, may help move these forward by enabling large-scale manufacturing.

In contrast, UIUC's Cunningham pointed out that standard approaches like PCR, ELISA, digital PCR, and Simoa "all have limitations due to the use of some combination of enzymes, thermal cycles, precise temperature control," and other inherent features.

Isothermal approaches, like loop-mediated isothermal amplification (LAMP), have issues with primer design and false positive amplification, and it is unclear how much commercial traction other isothermal NAAT methods — such as recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), exponential amplification reaction (EXPAR), or nucleic acid sequence-based amplification — are getting.

Despite these challenges, molecular technologies that use amplification currently remain the best option for most diagnostics developers.

"While conventional label-free biosensors like SPR, nanoresistors, acoustic sensors, and waveguides all can perform direct detection without the enzymes, their detection limits are still orders of magnitude too high to compete with PCR and digital PCR," Cunningham noted.

Driven by graphene

A recent review of graphene-based approaches noted that they can be enhanced with bioreceptors and combined with things like FETs and electrochemical biosensors to provide signals in the form of current or voltage changes, or with a readout such as surface plasmon resonance to provide signals by changes in SPR angle.

"The potential of graphene-based biosensors to detect infectious diseases is enormous, and the translation of this concept into clinical use would be revolutionary," according to a review of self-assembled biomolecules on graphene.

The accelerated popularity of electronic detection tech has been supported by recent advances in the manufacture of graphene — a hexagonal arrangement of single carbon atoms.

Synthesis of graphene is now more advanced, with innovations like roll-to-roll manufacturing allowing developers to purchase large sheets and fabricate highly reproducible sensors for a much lower cost than previous research-grade graphene devices, or ones that relied on silicon.

These new graphene approaches support some of the startups in the amplification-free molecular and single-molecule testing domains.

For example, a company called Flexotronix has developed high-speed, reel-to-reel manufacturing lines for printed electronics using conductive inks and proprietary lateral flow formats, funded in part by the Bill & Melinda Gates Foundation. The manufacturing line purportedly has the capacity to make up to 5 billion biosensor-based tests per year. 

Last June, Flexotronix partnered with GrapheneDx and Sapphiros to develop graphene biosensor-based diagnostic tests for POC and consumer settings. In January, OraSure made a $30 million investment in Sapphiros and entered an exclusive distribution deal for these diagnostic tests, noting at the time that there is a potential for the products to include molecular technologies in the future.

Graphene and other substrates can be functionalized with different molecules to create biosensors, or biomolecular sensors.

Cunningham's firm, Atzeyo Biosensors, uses a biosensor approach that can detect DNA, RNA, and protein biomarkers with gold nanotechnology and a method called photonic resonance absorption microscopy. The approach requires minimal sample prep, can be multiplexed, and yields recorded images to enable both qualitative and quantitative results.

Meanwhile, Ireland-based Altratech combines peptide nucleic acid, or PNA technology, along with beads, and complementary metal-oxide-semiconductors (CMOS) to measure capacitance for direct molecular testing. The low cost of this approach is evidenced by the fact that the firm previously stated it is aiming for prices below $30 for a reader device and less than $5 per test.

And, Scanogen's single-molecule tethering, or SMOLT, approach generates a signal when micron-sized beads tethered by double-stranded DNA probes are displaced in the presence of a target pathogen, with displacement detected using a low-magnification lens and a low-cost digital camera. The Baltimore-based firm has been developing this approach for direct-from-blood sepsis testing.

Other companies, like IdentifySensors Biologics and Cardea Bio, are also merging the electrical properties of newly mass-producible graphene with its ability to be easily functionalized. The resulting biosensors can capture pathogen nucleic acids and yield direct electrical readouts with high sensitivity.

Another electronic technique that is driving new startups is surface plasmon resonance. Using this technology, startups such as Nicoya Lifesciences analyze proteins, but the approach is reportedly compatible with nucleic acids and other molecules.

And, an alternate use of carbon is to create carbon fiber microelectrodes. These can be combined with fast-scan cyclic voltammetry to create a technique which can potentially be used for diagnostics. A similar microelectrode approach was among the core technologies of Xagenic, a 2009 amplification-free diagnostics startup, some of whose technology was ultimately acquired by General Atomics.

Non-molecular diagnostic uses for biosensors abound, as well. For example, Avails Medical deploys functionalized biosensors for phenotypic antimicrobial susceptibility testing. Affinity Biosensors uses them for bacteremia testing, while Diagmetrics uses them for breath-based respiratory testing, and GRIP Molecular uses them to detect antigens, antibodies, enzymes, hormones, peptides, and even small molecules. Indeed, this sector seems to be booming, with other startups commercializing biosensors for diagnostics including Glympse Bio, Hawkeye Bio, Nutromics, and Nanopath.

A focus on CRISPR

New CRISPR approaches on the horizon may also obviate the need for amplification in molecular diagnostics.

Cardea Bio uses a CRISPR-Cas9-based biosensor diagnostic device that uses a graphene transistor to analyze DNA in its native state, without the need for amplification, labeling, or optical instruments.

A Rice University and University of Connecticut research team created an "ultrasensitive" Cas13a-based COVID molecular diagnostic, and a Lab on a Chip study last year described a 25-minute amplification-free microfluidic chip-based SARS-CoV-2 test with a limit of detection of 10 attomolar.

And, a study earlier this month showed a method called autocatalytic Cas12a circular DNA amplification reaction (AutoCAR) could be applied for nucleic acid diagnostics to detect approximately 1 copy of DNA per microliter at room temperature without additional signal amplification.

Tempering this, a Lab on a Chip review from 2022 noted that amplification-free CRISPR approaches still face challenges with standardization, multiplexing, creating integrated platforms, and keeping costs low.

All of Sherlock Biosciences' molecular diagnostics in development require some type of nucleic acid amplification, according to Bryan Dechairo, the firm's CEO. Sherlock's COVID-19 test, which received Emergency Use Authorization from the FDA, used LAMP, but the firm is also developing a proprietary room-temperature amplification technology for use with a handheld device.

According to Dechairo, nucleic acid amplification is required for CRISPR-based human MDx approaches in order to reach the levels of clinical sensitivity required for regulatory clearance, especially for sample types like vaginal matrix.

The CRISPR-based diagnostics space has also seen a number of new entrants in the past two years, all of whom also use amplification in their workflows so far.

For example, startup VedaBio is developing an approach that uses proprietary engineered complexes it calls CRISPR-Cas ribonucleoproteins, or RNPs, to detect nucleic acids of interest and subsequently amplify signal.

With core technology based on research from Tulane University, newcomer Intelligenome is developing blood-based CRISPR testing for Mycoplasma tuberculosis. The firm's "lab in a tube" test contains high-fidelity DNA polymerase, PCR buffer with integrated double-stranded stabilization protein, dNTP, and primer pairs of a sequence of DNA that is unique to M. tuberculosis for nucleic acid amplification, according to the startup's website. It then adds the Cas enzyme and a reaction buffer mixed with fluorescent probes and gRNA and uses paper strips for accurate identification of M. tuberculosis using a portable fluorescent reader.

Despite the excitement over amplification-free detection, however, "the CRISPR work, while holding some promise for diagnostics, has gotten oversized attention, especially from those outside of the diagnostics field," according to Columbia's Sia.

Potential hurdles

So far, none of these newer technologies has been evaluated by the FDA, and without a predicate device, the path to regulatory clearance goes through the more challenging premarket approval pathway.

However, some of the component technologies of these tests are incorporated into a few FDA Emergency Use Authorized IVDs.

Sherlock Biosciences' COVID test was the first-ever CRISPR-based diagnostic assay to be reviewed by the FDA, for example, while Mammoth Biosciences obtained EUA for a high-throughput CRISPR-based test and Proof Diagnostics submitted point-of-care test.

Similarly, with support from RADx, Ellume obtained EUA for a test that uses quantum dots to enhance detection of an immunoassay. As Bruce Tromburg, director of the National Institute of Biomedical Imaging and Bioengineering, said previously, the idea that such a cutting-edge approach could be regulated for home use was unimaginable before the pandemic.

Now federally supported as an at-home test for visually impaired people, Ellume also exemplifies a technology whose increased adoption has been inspired in part by patient advocacy, an approach that might be useful to amplification-free startups.

Some FDA-cleared technologies do share attributes with these startups and could also serve as a point of reference for regulators.

The Thermo Fisher Scientific Quantigene Plex is a hybridization microarray in which binding of target nucleic acid sequences elicits amplification of a fluorescent signal rather than the mRNA. And the Roche Diagnostics Eplex is perhaps the most commercially advanced biosensing system, although its biosensing and electronic detection requires amplification as well.

In terms of Atzeyo Biosensors' technology, Cunningham said a comparable approach could be that of NanoString Technologies (now part of Bruker), a firm which uses non-amplified detection paired with fluorescently tagged DNA barcodes for counting and visualization. 

However, "compared to us, NanoString has a pretty complex workflow and expensive detection instrument, which is essentially a confocal fluorescence microscope," he said.

Competition in this space is also intensifying. During the pandemic, a multitude of companies launched point-of-care and handheld nucleic acid amplification systems and other diagnostics for infectious diseases, and many of these firms are now working to expand and diversify test menus.

More recently, a number of rapid POC molecular systems from larger players have been launched or disclosed — including BioMérieux's SpotFire, QuidelOrtho's Savanna, Becton Dickinson's Elience, or Bio-Rad Laboratories' PCR One — and these would also likely provide competition for direct detection approaches.

In the current at-home diagnostics market, broadly, firms are encountering a rocky path to reimbursement.

The fates of the EUA over-the-counter molecular tests, all of which use LAMP, also illustrate the challenges in this space. The recently launched Aptitude Medical Metrix device incorporates an integrated microfluidic electrochemical sensor and was the subject of an exclusive US distribution agreement with Sekisui earlier this year. The 3EO Health test was the subject of a $6 million RADx award last year, while the Lucira Health test was acquired by Pfizer in a bankruptcy action.

Three other manufacturers of instrumented rapid molecular systems, however, went public via IPO, then quickly saw stock prices plummet. Despite the systems being underpinned by vetted and validated LAMP technologies, the cost of the instruments and assays as well as other business issues led Talis Biomedical to enter a strategic review process after experiencing manufacturing problems, LumiraDx to be delisted, and Cue Health to shut down.

Even more broadly, emerging approaches in direct infectious disease testing are also likely to impact amplification-free MDx startups. Oxford Nanopore recently announced that it is developing a sequencing-based test for drug-resistant tuberculosis and is expected to roll it out in partnership with BioMérieux. Nanopore-based approaches have also recently shown promise for rapid respiratory infection testing in the ICU and are being deployed by startups Omixon and GenDx for HLA typing for transplant medicine applications.

Still, the potential to detect other biomarker types, especially proteins, is a distinct advantage of some amplification-free approaches over PCR, Schmidt said, and potentially also over sequencing-based approaches.

Nevertheless, he agreed that technological approaches that are good for the high-volume clinical lab might not be ideal for rapid tests, point-of-care tests, or research instruments.

"There are likely different best solutions based on the use case," Schmidt said.