October 4, 2023 | Soon it may be possible for people to walk into their doctor’s office anywhere in the world and learn from a fingerprick blood sample if they are genetically predisposed to developing osteoporosis. It will matter not if those individuals are anywhere near the age of menopause, or if they’re male or female, because the SNPs being measured were present from the day they were born and men are by no means immune to the condition, according to Ciara K. O’Sullivan, Ph.D., research professor at the Catalan Institute for Research and Advanced Studies at the University of Rovira i Virgili, a public university in Tarragona, Spain.
SNPs—single nucleotide polymorphism—can foretell the risk of bone fracture as well as a long list of unwelcome health conditions from malaria to cardiovascular disease using a generic platform under development by O’Sullivan and her collaborators across Europe. All that’s required to change applications is swapping out the letters (A, T, C, and G) in DNA sequences corresponding to the four different SNP types that get screen-printed as gold dots by the novel electrode array device.
The biosensor gives a risk score based on a combination of five previously identified SNPs that emerged as the most highly relevant for establishing the risk of developing osteoporosis, based on a genome-wide analysis of about 50,000 individuals whose family history and lifestyle choices were also known (Bone, DOI: 10.1016/j.bone.2010.01.001), says O’Sullivan. Since the DNA does not have to be purified from the blood, the analysis can be completed in about 15 minutes and at a cost of roughly 50 cents per SNP (reagent per electrode), as most recently reported in ACS Central Science (DOI: 10.1021/acscentsci.3c00243).
Rates of osteoporosis are “massively increasing” all over the world while awareness of the disease and its risk factors is shockingly low, O’Sullivan points out. From their first fracture, people’s quality of life tends to plummet and 25% of them die within a year. Knowing their predisposition to the condition is therefore crucial.
Early intervention is imperative to reducing the morbidity and mortality associated with the disease, which affects about 54 million people in the U.S., according to the International Osteoporosis Foundation. The most common technique used to measure changes in bone mineral density, dual-energy X-ray absorptiometry, is not sufficiently sensitive to detect the problem until a significant amount of damage has already happened.
Predisposition isn’t a diagnosis, O’Sullivan stresses, but some of the disease risk factors are modifiable—including smoking, excessive alcohol consumption, low calcium intake, and being sedentary. Given an early warning of their susceptibility, patients might adopt beneficial lifestyle changes early and start taking vitamin D and getting regular checkups with their doctor.
For now, the imagined clinical utility of the new device is limited to flagging at-risk individuals, says O’Sullivan. But given the flood of new genetic information that is emerging, at both the microRNA and transcriptome level, the platform may one day be deployed for diagnosing disease, she adds.
The device O’Sullivan and her team are developing has an electrode array to which DNA fragments for each SNP are attached. When lysed whole blood is applied to it, any DNA matching the SNPs binds the sequences and is amplified with recombinase polymerase (an alternative to PCR) that incorporates ferrocene to facilitate electrochemical detection.
Information about the SNPs comes from the heterozygosity and homozygosity of the markers, she explains. The electrode array is a simple device made by the same type of screen printing used to imprint images on T-shirts, except the ink—gold in this case—is being applied on a substrate made of ceramic rather than cotton fabric. Once the pattern has been created, the DNA fragments are applied.
Each SNP location in the genome can have up to four bases (A, C, G, and T), one for each nucleotide. While human DNA is virtually identical person to person, differences of only 0.1% represent millions of different locations where variation can occur that provide clues about predispositions, says O’Sullivan.
Literally millions of white blood cells can be found in a fingerprick of blood, which the researchers here are opening (lysing) via heating to let the DNA out. Two complementary single-stranded molecules will bond together if completely paired and, when they don’t, the outliers are signaled by no amplification on the electrode surfaces of the device.
For many years, O’Sullivan says, her focus was on developing electrochemical biosensors, the basis of the once-ubiquitous diabetic blood test strips that have been largely replaced by personal glucose monitors. But more recently she and her team have sharpened their focus on simplifying the testing process via lateral flow detection as well as multiplexing the process to many different biomarkers at the same time.
In fact, one of the big differences between this latest platform and real-time polymerase chain reaction (PCR) commonly used in clinical laboratories is scale, she explains. “If you are using real-time PCR, you can maybe do four different SNPs at the same time from one sample, whereas with our approach you can do as many as you want... and that has no impact on the time nor the [per SNP] cost.”
Although the research team looked only at SNPs for this initial study, continues O’Sullivan, the overall vision for the device is to examine different types of biomarkers—including potentially proteins and messenger RNA or microRNA—in a single fingerprick blood sample.
Other related projects now underway using the same electrode array include detection of 14 high-risk genotypes of the human papillomavirus virus (HPV) from a vaginal swab, designed to be a prescreening tool for women in developing countries who never received the HPV vaccine that helps prevent cervical cancer, she says. Individuals diagnosed with high-risk HPV could then be encouraged to return for further screening and to take preventive steps (e.g., stop smoking and avoid long-term use of birth control pills) or get a curative treatment if precancerous cells are found.
The platform is at the same time being investigated for its potential in triaging patients presenting with signs of a bacterial infection using a spot of their blood, says O’Sullivan. In this case, the device can both rapidly identify the disease (e.g., malaria or HIV) as well allowing identification of antibiotic resistance and thus a correct choice of the medicines that can kill the pathogens or stop their growth—a key feature given the growing problem of antibiotic resistance in both the developing and developed world.
In the case of tuberculosis, a sputum sample might instead be used to identify the correct antibiotic to give individuals, she says. Not only would that save the lives of those patients, but also prevent them from inadvertently spreading the disease because the wrong antibiotic was chosen.
Sputum is a bit more difficult to work with than blood and requires a more complicated lysing process, says O’Sullivan, although many different DNA extraction methods are available. With the cervical samples, a surfactant is generally used that opens the cells in a matter of seconds.
At present, the biosensor platform is being used purely for research purposes, O’Sullivan says. The path to commercialization, as it plays out in Europe, begins with a consortium of partners—which in this case initially incudes Labman Automation (U.K.), which is developing the instrumentation; Fraunhhofer-Institute für Microtechnik Mainz (Germany) and MicroLIQUID (Spain), which are developing microsystems; and the University of Gent (Belgium), which is helping design the DNA primers for use on the electrodes as well as developing sensors for the detection of protein bone turnover markers.
Clinical validation for the osteoporosis indication will be done on several hundred patients whose biobanked blood samples have already been analyzed via real-time PCR, thanks to a partnership with the Medical University of Graz in Austria, she notes. A comparatively expensive and complex gold-standard lysis method was used on the blood cells to extract and purify the DNA.
Although clinical use may begin at the Medical University of Graz, the aim is to widely implement the device across both the developed and developing world, says O’Sullivan. Whatever diagnostic is ultimately deployed will need to be highly cost-effective, and thus easily adopted by physician offices, clinics, and hospitals.
Since the platform is “completely generic,” adds O’Sullivan, it could be applied to a whole range of conditions. She and her team just started a new project that is looking at both SNPs and proteins in a fingerprick of blood to assess predisposition risk for developing cardiovascular disease. “There is a whole range of diseases that can be detected at an early... enough [stage] to intervene and treat them correctly” in a personalized fashion based on pharmacogenomics, she notes.