Approach involves converting the concentration of sensor-bound target into a measurable glucose concentration.
Researchers have exploited the simplicity of personal glucose meters (PGMs) used by diabetics worldwide to develop a prototype platform for detecting and quantifying nonglucose targets such as recreational drugs, disease-related and other biological markers, or toxic substances in blood or serum samples. The technique developed by researchers at the University of Illinois at Urbana-Champaign is based on invertase-conjugated functional DNA sensors that are immobilized on magnetic beads. The approach in essence converts target detection by the DNA sensor into an easily readable glucose concentration, which is directly proportional to the amount of target present in the original sample.
Writing in Nature Chemistry, Yu Xiang, Ph.D., and Yi Lu, Ph.D., describe use of the approach to detect cocaine, interferon, adenosine, and uranium. They admit that the technique will need simplifying and automating for widespread use, as it currently requires two steps. However, they don’t foresee this as a major issue, “just as glucose meters have been developed from complicated devices into pocket-sized meters over 30 years of progress,” they point out. The researchers report on their developments in a paper titled “Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets.”
The wide success of PGMs is largely due to their portable size, low cost, reliable quantitative results, and simple operation, Drs. Xiang and Lu note. Although a number of methods have been developed to quantitatively detect other targets of interest using simple instruments, most of these devices have not been able to match the PGM in terms of wide commercial availability to the public.
In order to use existing PGMs for quantifying nonglucose targets, a method was required to equate glucose detection with the detection of other targets without changing design of the PGM itself. The researchers came up with the idea of using functional-DNA-conjugated to invertase, which would allow a concentration of glucose to serve as a quantitative read-out for other targets of interest present in a sample.
Functional DNAs include DNAzymes (also called deoxyribozymes, catalytic DNAs, or DNA enzymes) that act as catalysts, aptamers (that selectively bind targets), and aptazymes (which are a combination of the two). Functional DNAs and functional RNAs to a wide range of biological, biochemical, and chemical targets can be selected from libraries using in vitro selection, or the systematic evolution of ligands by exponential enrichment (SELEX).
Conjugating the DNA sensor to invertase (β-fructofuranosidase) provided the means to convert target-functional DNA binding into a glucose signal that could then be quantified using PGMs. Invertase is an enzyme that catalyzes the hydrolysis of sucrose into glucose, which can be detected by the PGM. And because even nanomolar levels of invertase are capable of converting a millimolar concentration of sucrose into glucose under ambient conditions, the enzyme represents an ideal catalyst for the amplification of “turn-on” signals in the PGM, the researchers stress.
In order to establish a direct relationship between target concentration and invertase concentration, the researchers immobilized the DNA sensor-invertase conjugates onto magnetic beads using streptavidin-biotin binding. Dissociation of the invertase-conjugated sensors into solution only occurs on binding of the DNA sensor moiety to its target in the sample.
The overall process essentially converts one signal into another. Interaction of the functional DNA portion of the invertase conjugate to its target in a blood sample causes release of the whole functional DNA-invertase conjugate from the magnetic beads into solution. The beads carrying unbound conjugates are then removed by a magnet. The invertase in solution then catalyzes the hydrolysis of sucrose into glucose, which can be quantified by the PGM. The quantity of invertase is thus directly proportional to the amount of target in the sample: the more target in the sample, the more DNA-invertase conjugate is released into solution from the beads, and the more glucose is produced by invertase activity on sucrose.
The researchers used the platform to develop invertase-conjugated aptamer sensors to measure cocaine, adenosine, and IFN-γ in serum and blood, and an invertase-conjugated functional DNAzyme-based sensor for detecting uranium. Target-induced release of the invertase-sensor conjugates from the magnetic beads on target binding occurred either due to structure switching of aptamers, or substrate cleavage by DNAzymes. The results showed that for each type of DNA sensor generated, the detection limit was 3.4 μM for cocaine, 18 μM for adenosine, 2.6 nM for interferon-γ, and 9.1 nM for uranium.
“Given the wide availability of PGMs and the ability of functional DNAs to recognize many targets, the method demonstrated here has the potential to change environmental monitoring and medical diagnostics for the public at home and in the field,” the authors conclude. “As more functional DNAs and RNAs are available or can be obtained through in vitro selection or SELEX to bind a broad range of targets, the method developed here can be used to quantify many other targets that functional DNAs and RNAs can recognize.”
They team’s next stage will be to simplify the overall process, as two steps are currently required to generate the glucose that can be read by the PGM. “We are working on integrating the procedures into one step to make it even simpler,” remarks Dr. Lu. “Our technology is new, and given time, it will be developed into an even more user-friendly format. The advantages of our method are high portability, low cost, wide availability, and quantitative detection of a broad range of targets in medical diagnostics and environmental monitoring. It is simple enough for someone to use at home without the high costs and long waiting period of going to the clinic or sending samples to professional labs.”